Ionizable lipid compounds, lipid nanoparticles comprising same and therapeutic uses thereof

ABSTRACT

The present invention relates to lipid nanoparticles for in vivo drug delivery and uses thereof, and the lipid nanoparticle are liver tissue-specific, have excellent biocompatibility and can deliver a gene therapeutic agent with high efficiency, and thus it can be usefully used in related technical fields such as lipid nanoparticle mediated gene therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Application No. 17/504,785filed on Oct. 19, 2021, which is a national phase of InternationalApplication No. PCT/KR2020/019502 filed on Dec. 31, 2020, which claimspriority to Korean Application No. 10-2020-0040586 filed on Apr. 2, 2020and Korean Application No. 10-2020-0005642 filed on Jan. 15, 2020, whichapplications are incorporated herein by reference.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Nov. 17, 2022, isnamed ENC-P30001C.xml and is 26,096 bytes in size.

TECHNICAL FIELD

The present invention relates to lipid nanoparticles for in vivo drugdelivery and uses thereof.

BACKGROUND ART

In the pharmaceutical formulation industry, the drug delivery system(DDS), designed to efficiently deliver the required amount of the drugby reducing side effects of the drug and maximizing efficacy andeffects, is a high value-added core technology which can create economicbenefits comparable to that of new drug development and has greatpotential for success and its purpose is to improve the quality ofpatient treatment by making drug administration more efficient.

The solubilization technology of poorly soluble drugs belonging to thedrug absorption promotion technology, which is one of the coretechnologies of the drug delivery system, is considered the mostreasonable way to reduce the development cost of new drug substances andat the same time increase the added value of currently marketed drugs.In particular, the development of improved new drugs through thedevelopment of drug solubilization technology in a situation where newdrug development conditions are poor as in Korea is a field that cancreate enormous added value at a low cost.

Gene therapy using a genetic drug delivery system is established in alarge hope of modifying genetic binding and to treat numerous diseases.In the successful and securely performing this gene therapy, theeffective gene delivery is one of the main challenges and the virusdelivery system was proved to be effective in gene delivery. However,due to some defects such as immunogenicity, limitation of the insertedDNA size and difficulties of mass production, the use of viruses arelimited as a gene delivery system. Non-viral gene carriers such ascationic liposome and polymers began to be noted as an alternative meansof a viral system.

Improved stability profile and ease of manufacturing and operation ofthe polymer delivery system triggered studies on the design andsynthesis of a non-toxic and biodegradable polymer carrier for effectiveand safe gene delivery. Poly(L-lysine), polyethylenimine, starburst,polyamidoamine dendrimer, and cationic liposome voluntarily, and thelike can be self-assembled and compress plasmid DNA (pDNA) into a smallstructure sufficiently to enter cells through endocytosis, and thereforethey have been widely studied as a non-viral gene delivery system.

Nucleic acids such as antisense RNA, siRNA, and the like are a materialcapable of inhibiting expression of specific proteins in vivo, and arespotlighted as an important tool for treatment of cancer, geneticdiseases, infectious diseases, autoimmune diseases, and the like (Novinaand Sharp, Nature, 430, 161-164, 2004). However, nucleic acids such assiRNA are difficult to deliver directly into cells and they are easilydecomposed by enzymes in the blood, so there are many studies toovercome them. To date, the method for delivering nucleic acids intocells, a method for carrying by mixing with a positive charge lipid orpolymer (named lipid-DNA conjugate (lipoplex) and polymer-DNA conjugate(polyplex), respectively) is mainly used (Hirko et al., Curr. Med.Chem., 10, 1185-93, 2003; Merdan et al., Adv. Drug. Deliv. Rev., 54,715-58, 2002). The lipid-DNA conjugate is combined with the nucleic acidto deliver the nucleic acid well to cells and thus it is used a lot atthe cell level, but in vivo, when injecting locally, in many cases, ithas a disadvantage of inducing inflammation in the body (Filonand andPhillips, Biochim Biophys. Acta, 1329, 345-56, 1997).

In addition, such a non-viral delivery system has a problem of lowtransfection efficiency. Many efforts have been tilted to enhancetransfection efficiency, but this is still far from the system that isstable. In addition, the carrier of the non-viral gene delivery systemrepresents a significantly high cytotoxicity due to poorbiocompatibility and non-biodegradability.

Under such a technical background, as the result that the presentinventors have tried to develop a novel particle which has excellentencapsulation efficiency and can effectively deliver an anionic drug, anucleic acid, and the like to a targeted organ or cell, they havecompleted the present invention by producing a lipid nanoparticlecomprising an ionizable lipid; phospholipid; cholesterol; and alipid-PEG (polyethyleneglycol) conjugate, and confirming that the lipidnanoparticle is delivered specifically to liver tissue and the drug suchas anionic compound or nucleic acid can be encapsulated with highefficiency.

DISCLOSURE Technical Problem

An object of the present invention is to provide a lipid nanoparticlecomprising an ionizable lipid in which a 6-membered heterocyclic amineand an alkyl-epoxide are boned, a phospholipid, cholesterol and alipid-PEG (polyethyleneglycol) conjugate.

Another object of the present invention is to provide a composition fordelivering a drug (anionic drug, nucleic acid or a combination thereof)comprising (1) the lipid nanoparticle; and (2) an anionic drug, anucleic acid, or a combination thereof.

Other object of the present invention is to provide a pharmaceuticalcomposition for preventing or treating liver disease comprising (1) thelipid nanoparticle; and (2) an anionic drug, a nucleic acid, or acombination thereof.

Technical Solution

This will be described in detail as follows. On the other hand, eachdescription and embodiment disclosed in the present invention may beapplied to each other description and embodiment. In other words, allcombinations of various elements disclosed herein fall within the scopeof the present invention. In addition, it cannot be considered that thescope of the present invention is limited by the specific descriptiondescribed below.

One aspect to achieve the above object provides a lipid nanoparticlecomprising an ionizable lipid in which a 6-membered heterocyclic amineand alkyl-epoxide are combined; phospholipid; cholesterol; and alipid-PEG (polyethyleneglycol) conjugate.

The lipid nanoparticle according to one example is liver tissue-specificand has excellent biocompatibility and can deliver a gene therapeuticagent and the like with high efficiency, and thus it can be usefullyused in related technical fields such as lipid nanoparticle-mediatedgene therapy and image diagnosis technology.

Herein, ‘ionizable lipid’ or ‘lipidoid’ mean an amine-containing lipidwhich can be easily protonated, and for example, it may be a lipid ofwhich charge state changes depending on the surrounding pH. Theionizable lipid may be one in which a 6-membered heterocyclic amine andalkyl-epoxide are combined. Specifically, the ionizable lipid may be acompound having characteristics similar to the lipid produced byreaction of the 6-membered heterocyclic amine and alkyl-epoxide, andmore specifically, it may be a compound produced by ring openingreaction of epoxide by reacting the 6-membered heterocyclic amine withalkyl-epoxide.

In one example, the ionizable lipid may be one in which a 6-memberedheterocyclic amine and alkyl-epoxide are combined by reacting them at amolar ratio of l:n (n=number of primary amines comprised in 6-memberedheterocyclic aminex2+number of secondary aminesxl). According to onespecific example, it may be prepared by mixing 246 amine and1,2-epoxydodecane at a molar ratio of 1:4 and reacting them under theconditions of 700 to 800 rpm and 85 to 95 for 2 to 4 days.

The ionizable lipid may be protonated (positively charged) at a pH belowthe pKa of a cationic lipid, and it may be substantially neutral at a pHover the pKa. In one example, the lipid nanoparticle may comprise aprotonated ionizable lipid and/or an ionizable lipid showing neutrality.

The ionizable lipid is an ionizable compound having characteristicssimilar to the lipid, and through electrostatic interaction with a drug(for example, anionic drug and/or nucleic acid), may play a role ofencapsulating the drug within the lipid nanoparticle with highefficiency.

The 6-membered heterocyclic amine may comprise a structure of piperazineor piperidine.

The 6-membered heterocyclic amine may be a chain or non-chain aminecomprising a tertiary amine, and according to one example, it may be oneor more kinds selected from the group consisting of

In one example, the 6-membered heterocyclic amine may be one or morekinds selected from the group consisting of1,4-bis(3-aminopropyl)piperazine, N-(3-Aminopropyl)piperidine,(1-Methyl-4-piperidinyl)methanamine,2-(4-Methyl-piperazin-1-yl)-ethylamine, 1-(2-Aminoethyl)piperazine, and1-(3-aminopropyl)piperazine.

According to the type of the amine comprised in the ionizable lipid, (i)the drug encapsulation efficiency, (ii) PDI (polydispersity index).and/or (iii) the drug delivery efficiency to liver tissue and/or cellsconstituting liver (for example, hepatocyte) and/or LSEC (liversinusoidal endothelial cell) of the lipid nanoparticle may be different.

The lipid nanoparticle comprising an ionizable lipid comprising an aminemay have one or more kinds of the following characteristics:

-   (1) encapsulating a drug with high efficiency;-   (2) uniform size of prepared particles (or having a low PDI value);    and/or-   (3) excellent drug delivery efficiency to liver tissue, and/or cells    constituting liver (for example, hepatocyte and/or LSEC).

According to one example, a lipid nanoparticle comprising an ionizablelipid comprising 1,4-bis(3-aminopropyl)piperazine (for example, Cas Nos.7209-38-3) may have one or more kinds of the following characteristicsthan a lipid nanoparticle comprising an ionizable lipid comprising othertypes of amines:

-   (1) encapsulating a drug with high efficiency;-   (2) uniform size of prepared particles (or having a low PDI value);    and/or-   (3) excellent drug delivery efficiency to liver tissue, and/or cells    constituting liver (for example, hepatocyte and/or LSEC).

The alkyl-epoxide may have the structure of Chemical formula 1 below.

The alkyl-epoxide may have a carbon length of C6 to C14, C6 to C12, C6to C10, C8 to C14, C8 to C12, C8 to C10, C10 to C14, C10 to C12, or C10,and for example, it may be 1,2-epoxydodecane of C10. By setting thecarbon number of the alkyl-epoxide comprised in the ionizable lipid tothe above range, it is possible to represent a high encapsulationefficiency for the drug encapsulated in the lipid nanoparticle.

In one example, the ionizable lipid may have the general formula ofChemical formula 2 below.

The structure of Chemical formula 2 is one example of the structure ofthe ionizable lipid according to one example, and the structure of theionizable lipid may be different depending on the type of the 6-memberedheterocyclic amine and alkyl-epoxide.

According to one example, a lipid nanoparticle comprising an ionizablelipid having the structure of Chemical formula 2 may have one or morekinds of the following characteristics than a lipid nanoparticlecomprising other types of ionizable lipids:

-   (1) encapsulating a drug with high efficiency;-   (2) uniform size of prepared particles (or having a low PDI value);    and/or-   (3) excellent drug delivery efficiency to liver tissue, and/or cells    constituting liver (for example, hepatocyte and/or LSEC).

According to one example, the lipid nanoparticle may have a pKa of 5 to8, 5.5 to 7.5, 6 to 7, or 6.5 to 7. The pKa is an acid dissociationconstant, and refers to what is generally used as an index indicatingthe acid strength of a target substance. The pKa value of the lipidnanoparticle is important in terms of in vivo stability of the lipidnanoparticle and drug release of the lipid nanoparticle. In one example,the lipid nanoparticle showing the pKa value in the above range may besafely delivered to a target organ (for example, liver) and/or targetcell (hepatocyte, and/or LSEC) in vivo, and reach to the target organand/or target cell, and after endocytosis, exhibit a positive charge torelease an encapsulated drug through electrostatic interaction with ananionic protein of the endosome membrane.

The phospholipid of the elements of the lipid nanoparticle according toone example plays a role of covering and protecting a core formed byinteraction of the ionizable lipid and drug in the lipid nanoparticle,and may facilitate cell membrane permeation and endosomal escape duringintracellular delivery of the drug by binding to the phospholipidbilayer of a target cell.

For the phospholipid, a phospholipid which can promote fusion of thelipid nanoparticle according to one example may be used withoutlimitation, and for example, it may be one or more kinds selected fromthe group consisting of dioleoylphosphatidylethanolamine (DOPE),distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine(POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), distearoylphosphatidylethanolamine (DSPE),phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine(POPE),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC),1,2-dioleoyl-sn-glycero-3-[phospho-L-serine](DOPS),1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] and the like. In oneexample, the lipid nanoparticle comprising DOPE may be effective in mRNAdelivery (excellent drug delivery efficiency to mRNA), and in otherexample, the lipid nanoparticle comprising DSPE may be effective insiRNA delivery (excellent drug delivery efficiency to siRNA).

The cholesterol of the elements of the lipid nanoparticle according toone example may provide morphological rigidity to lipid filling in thelipid nanoparticle and be dispersed in the core and surface of thenanoparticle to improve the stability of the nanoparticle.

Herein, “lipid-PEG (polyethyleneglycol) conjugate”, “lipid-PEG”,“PEG-lipid”, “PEG-lipid”, or “lipid-PEG” refers to a form in which lipidand PEG are conjugated, and means a lipid in which a polyethylene glycol(PEG) polymer which is a hydrophilic polymer is bound to one end. Thelipid-PEG conjugate contributes to the particle stability in serum ofthe nanoparticle within the lipid nanoparticle, and plays a role ofpreventing aggregation between nanoparticles. In addition, the lipid-PEGconjugate may protect nucleic acids from degrading enzyme during in vivodelivery of the nucleic acids and enhance the stability of nucleic acidsin vivo and increase the half-life of the drug encapsulated in thenanoparticle.

In the lipid-PEG conjugate, the PEG may be directly conjugated to thelipid or linked to the lipid via a linker moiety. Any linker moietysuitable for binding PEG to the lipid may be used, and for example,includes an ester-free linker moiety and an ester-containing linkermoiety. The ester-free linker moiety includes not only amido (—C(O)NH—),amino (—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea(—NHC(O)NH—), disulfide (—S—S—), ether (—O—), succinyl(—(O)CCH2CH2C(O)—), succinamidyl (—NHC(O)CH2CH2C(O)NH—), ether,disulfide but also combinations thereof (for example, a linkercontaining both a carbamate linker moiety and an amido linker moiety),but not limited thereto. The ester-containing linker moiety includes forexample, carbonate (—OC(O)O—), succinoyl, phosphate ester (—O—(O)POH—O—), sulfonate ester, and combinations thereof, but not limitedthereto.

In one example, the average molecular weight of the lipid-PEG conjugatemay be 100 daltons to 10,000 daltons, 200 daltons to 8,000 daltons, 500daltons to 5,000 daltons, 1,000 daltons to 3,000 daltons, 1,000 daltonsto 2,600 daltons, 1,500 daltons to 2,600 daltons, 1,500 daltons to 2,500daltons, 2,000 daltons to 2,600 daltons, 2,000 daltons to 2,500 daltons,or 2,000 daltons.

For the lipid in the lipid-PEG conjugate, any lipid capable of bindingto polyethyleneglycol may be used without limitation, and thephospholipid and/or cholesterol which are other elements of the lipidnanoparticle may be also used. Specifically, the lipid in the lipid-PEGconjugate may be ceramide, dimyristoylglycerol (DMG),succinoyl-diacylglycerol (s-DAG), distearoylphosphatidylcholine (DSPC),distearoylphosphatidylethanolamine (DSPE), or cholesterol, but notlimited thereto.

In one example, the lipid-PEG conjugate may be PEG bound todialkyloxypropyl (PEG-DAA), PEG bound to diacylglycerol (PEG-DAG), PEGbound to phospholipid such as phosphatidylethanolamine (PEG-PE), PEGconjugated to ceramide (PEG-CER, ceramide-PEG conjugate, ceramide-PEG,PEG-ceramide conjugate or PEG-ceramide), cholesterol or PEG conjugatedto derivative thereof, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE,PEG-DPPC, PEG-DSPE(DSPE-PEG), and a mixture thereof, and for example,may be C 16-PEG2000 ceramide(N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethyleneglycol)2000]}), DMG-PEG 2000, 14:0 PEG2000 PE.

According to one example, the case of comprising a lipid nanoparticlecomprising a ceramide-PEG conjugate may have one or more kinds of thefollowing characteristics than the case of comprising a lipidnanoparticle comprising other types of lipid-PEG conjugates:

-   (1) encapsulating a drug with high efficiency;-   (2) uniform size of prepared particles (or having a low PDI value);    and/or-   (3) excellent drug delivery efficiency to liver tissue, and/or cells    constituting liver (for example, hepatocyte and/or LSEC).

The PEG in the lipid-PEG conjugate is a hydrophilic polymer and has anability to inhibit adsorption of serum proteins, and increases thecirculation time of lipid nanoparticles in the body and can play a roleof preventing aggregation between nanoparticles. In addition, thelipid-PEG conjugate may exhibit a stealth function in vivo to preventdegradation of nanoparticles.

The PEG may be what a functional group binds to a side not bound to alipid (functionalized PEG). In this case, the functional group that canbe used may be one or more kinds selected from the group consisting ofsuccinyl group, carboxylic acid, maleimide, amine group, biotin, cyanurgroup and folate, and the like.

According to one example, the lipid-PEG conjugate may be comprised inthe lipid nanoparticle in an amount of 0.1 to 15 mol %, 0.25 to 15 mol%, 0.5 to 15 mol %, 1 to 15 mol %, 1.5 to 15 mol %, 2 to 15 mol %, 2.5to 15 mol %, 0.1 to 12.5 mol %, 0.25 to 12.5 mol %, 0.5 to 12.5 mol %, 1to 12.5 mol %, 1.5 to 12.5 mol %, 2 to 12.5 mol %, 2.5 to 12.5 mol %,0.1 to 10 mol %, 0.25 to 10 mol %, 0.5 to 10 mol %, 1 to 10 mol %, 1.5to 10 mol %, 2 to 10 mol %, 2.5 to 10 mol %, 0.1 to 7.5 mol %, 0.25 to7.5 mol %, 0.5 to 7.5 mol %, 1 to 7.5 mol %, 1.5 to 7.5 mol %, 2 to 7.5mol %, 2.5 to 7.5 mol %, 0.1 to 5 mol %, 0.25 to 5 mol %, 0.5 to 5 mol%, 1 to 5 mol %, 1.5 to 5 mol %, 2 to 5 mol %, 2.5 to 5 mol %, 0.1 to 3mol %, 0.25 to 3 mol %, 0.5 to 3 mol %, 1 to 3 mol %, 1.5 to 3 mol %, 2to 3 mol %, 2.5 to 3 mol %, 0.1 to 2.5 mol %, 0.25 to 2.5 mol %, 0.5 to2.5 mol %, 1 to 2.5 mol %, 1.5 to 2.5 mol %, 2 to 2.5 mol %, 0.1 to 4.5mol %, 0.25 to 4.5 mol %, 0.5 to 4.5 mol %, 1 to 4.5 mol %, 1.5 to 4.5mol %, 2 to 4.5 mol %, 2.5 to 4.5 mol %, 0.1 to 4 mol %, 0.25 to 4 mol%, 0.5 to 4 mol %, 1 to 4 mol %, 1.5 to 4 mol %, 2 to 4 mol %, 2.5 to 4mol %, 0.1 to 3.5 mol %, 0.25 to 3.5 mol %, 0.5 to 3.5 mol %, 1 to 3.5mol %, 1.5 to 3.5 mol %, 2 to 3.5 mol %, 2.5 to 3.5 mol %, 0.1 to 2.0mol %, 0.1 to 1.5 mol %, 0.1 to 1.0 mol %, 0.5 to 2.0 mol %, 0.5 to 1.5mol %, 0.5 to 1.0 mol %, 1.0 to 2.0 mol %, 1.0 to 1.5 mol %, 1.5 to 2.0mol %, 1.0 mol %, or 1.5 mol %.

In one example, the liver tissue, hepatocytes and/or LSEC targetingeffect (drug delivery effect) of the lipid nanoparticle may be dependenton the content of the lipid-PEG conjugate comprised in the lipidnanoparticle.

For example, the lipid nanoparticle comprising the lipid-PEG conjugatein an amount of 0.1 to 15 mol %, 0.25 to 15 mol %, 0.5 to 15 mol %, 1 to15 mol %, 1.5 to 15 mol %, 2 to 15 mol %, 2.5 to 15 mol %, 0.1 to 12.5mol %, 0.25 to 12.5 mol %, 0.5 to 12.5 mol %, 1 to 12.5 mol %, 1.5 to12.5 mol %, 2 to 12.5 mol %, 2.5 to 12.5 mol %, 0.1 to 10 mol %, 0.25 to10 mol %, 0.5 to 10 mol %, 1 to 10 mol %, 1.5 to 10 mol %, 2 to 10 mol%, 2.5 to 10 mol %, 0.1 to 7.5 mol %, 0.25 to 7.5 mol %, 0.5 to 7.5 mol%, 1 to 7.5 mol %, 1.5 to 7.5 mol %, 2 to 7.5 mol %, 2.5 to 7.5 mol %,0.1 to 5 mol %, 0.25 to 5 mol %, 0.5 to 5 mol %, 1 to 5 mol %, 1.5 to 5mol %, 2 to 5 mol %, 2.5 to 5 mol %, 0.1 to 3 mol %, 0.25 to 3 mol %,0.5 to 3 mol %, 1 to 3 mol %, 1.5 to 3 mol %, 2 to 3 mol %, 2.5 to 3 mol%, 0.1 to 2.5 mol %, 0.25 to 2.5 mol %, 0.5 to 2.5 mol %, 1 to 2.5 mol%, 1.5 to 2.5 mol %, 2 to 2.5 mol %, 0.1 to 4.5 mol %, 0.25 to 4.5 mol%, 0.5 to 4.5 mol %, 1 to 4.5 mol %, 1.5 to 4.5 mol %, 2 to 4.5 mol %,2.5 to 4.5 mol %, 0.1 to 4 mol %, 0.25 to 4 mol %, 0.5 to 4 mol %, 1 to4 mol %, 1.5 to 4 mol %, 2 to 4 mol %, 2.5 to 4 mol %, 0.1 to 3.5 mol %,0.25 to 3.5 mol %, 0.5 to 3.5 mol %, 1 to 3.5 mol %, 1.5 to 3.5 mol %, 2to 3.5 mol %, 2.5 to 3.5 mol %, 0.1 to 10 mol %, 0.1 to 5.0 mol %, 0.1to 4.5 mol %, 0.1 to 4.0 mol %, 0.1 to 3.5 mol %, 0.1 to 3.0 mol %, 0.1to 2.5 mol %, 0.1 to 2.0 mol %, 0.1 to 1.5 mol %, 0.1 to 1.0 mol %, 0.5to 10 mol %, 0.5 to 5.0 mol %, 0.5 to 4.5 mol %, 0.5 to 4.0 mol %, 0.5to 3.5 mol %, 0.5 to 3.0 mol %, 0.5 to 2.5 mol %, 0.5 to 2.0 mol %, 0.5to 1.5 mol %, 0.5 to 1.0 mol %, 1.0 to 10 mol %, 1.0 to 5.0 mol %, 1.0to 4.5 mol %, 1.0 to 4.0 mol %, 1.0 to 3.5 mol %, 1.0 to 3.0 mol %, 1.0to 2.5 mol %, 1.0 to 2.0 mol %, 1.0 to 1.5 mol %, 1.5 to 10 mol %, 1.5to 5.0 mol %, 1.5 to 4.5 mol %, 1.5 to 4.0 mol %, 1.5 to 3.5 mol %, 1.5to 3.0 mol %, 1.5 to 2.5 mol %, 1.5 to 2.0 mol %, 1.0 mol %, or 1.5 mol% (than the lipid nanoparticle comprising the lipid-PEG conjugate in acontent outside the above range) may have an excellent targeting effectto hepatocytes.

As another example, the lipid nanoparticle comprising the lipid-PEGconjugate in an amount of 0.1 to 15 mol %, 0.25 to 15 mol %, 0.5 to 15mol %, 1 to 15 mol %, 1.5 to 15 mol %, 2 to 15 mol %, 2.5 to 15 mol %,0.1 to 12.5 mol %, 0.25 to 12.5 mol %, 0.5 to 12.5 mol %, 1 to 12.5 mol%, 1.5 to 12.5 mol %, 2 to 12.5 mol %, 2.5 to 12.5 mol %, 0.1 to 10 mol%, 0.25 to 10 mol %, 0.5 to 10 mol %, 1 to 10 mol %, 1.5 to 10 mol %, 2to 10 mol %, 2.5 to 10 mol %, 0.1 to 7.5 mol %, 0.25 to 7.5 mol %, 0.5to 7.5 mol %, 1 to 7.5 mol %, 1.5 to 7.5 mol %, 2 to 7.5 mol %, 2.5 to7.5 mol %, 0.1 to 5 mol %, 0.25 to 5 mol %, 0.5 to 5 mol %, 1 to 5 mol%, 1.5 to 5 mol %, 2 to 5 mol %, 2.5 to 5 mol %, 0.1 to 3 mol %, 0.25 to3 mol %, 0.5 to 3 mol %, 1 to 3 mol %, 1.5 to 3 mol %, 2 to 3 mol %, 2.5to 3 mol %, 0.1 to 2.5 mol %, 0.25 to 2.5 mol %, 0.5 to 2.5 mol %, 1 to2.5 mol %, 1.5 to 2.5 mol %, 2 to 2.5 mol %, 0.1 to 4.5 mol %, 0.25 to4.5 mol %, 0.5 to 4.5 mol %, 1 to 4.5 mol %, 1.5 to 4.5 mol %, 2 to 4.5mol %, 2.5 to 4.5 mol %, 0.1 to 4 mol %, 0.25 to 4 mol %, 0.5 to 4 mol%, 1 to 4 mol %, 1.5 to 4 mol %, 2 to 4 mol %, 2.5 to 4 mol %, 0.1 to3.5 mol %, 0.25 to 3.5 mol %, 0.5 to 3.5 mol %, 1 to 3.5 mol %, 1.5 to3.5 mol %, 2 to 3.5 mol %, 2.5 to 3.5 mol %, 0.1 to 10 mol %, 0.1 to 5.0mol %, 0.1 to 4.5 mol %, 0.1 to 4.0 mol %, 0.1 to 3.5 mol %, 0.1 to 3.0mol %, 0.1 to 2.5 mol %, 0.1 to 2.0 mol %, 0.1 to 1.5 mol %, 0.1 to 1.0mol %, 0.5 to 10 mol %, 0.5 to 5.0 mol %, 0.5 to 4.5 mol %, 0.5 to 4.0mol %, 0.5 to 3.5 mol %, 0.5 to 3.0 mol %, 0.5 to 2.5 mol %, 0.5 to 2.0mol %, 0.5 to 1.5 mol %, 0.5 to 1.0 mol %, 1.0 to 10 mol %, 1.0 to 5.0mol %, 1.0 to 4.5 mol %, 1.0 to 4.0 mol %, 1.0 to 3.5 mol %, 1.0 to 3.0mol %, 1.0 to 2.5 mol %, 1.0 to 2.0 mol %, 1.0 to 1.5 mol %, 1.5 to 10mol %, 1.5 to 5.0 mol %, 1.5 to 4.5 mol %, 1.5 to 4.0 mol %, 1.5 to 3.5mol %, 1.5 to 3.0 mol %, 1.5 to 2.5 mol %, 1.5 to 2.0 mol %, 1.0 mol %,or 1.5 mol % (than the lipid nanoparticle comprising the lipid-PEGconjugate in a content outside the above range) may have an excellenttargeting effect to LSEC.

According to one example, the cholesterol may be comprised in the lipidnanoparticle in an amount of 10 to 60 mol %, 20 to 60 mol %, 30 to 60mol %, 30 to 55 mol %, 30 to 52.5 mol %, 30 to 52 mol %, 30 to 51 mol %,30 to 50 mol %, 30 to 47.5 mol %, 30 to 45 mol %, 30 to 44 mol %, 30 to43.5 mol %, 30 to 43 mol %, 30 to 41.5 mol %, 30 to 40 mol %, 30 to 39.5mol %, 35 to 60 mol %, 35 to 55 mol %, 35 to 52.5 mol %, 35 to 52 mol %,35 to 51 mol %, 35 to 50 mol %, 35 to 47.5 mol %, 35 to 45 mol %, 35 to44 mol %, 35 to 43.5 mol %, 35 to 43 mol %, 35 to 41.5 mol %, 35 to 40mol %, 35 to 39.5 mol %, 37 to 60 mol %, 37 to 55 mol %, 37 to 52.5 mol%, 37 to 52 mol %, 37 to 51 mol %, 37.5 to 50 mol %, 37.5 to 47.5 mol %,37.5 to 45 mol %, 37.5 to 44 mol %, 37.5 to 43.5 mol %, 37.5 to 43 mol%, 37.5 to 41.5 mol %, 37.5 to 40 mol %, 37.5 to 39.5 mol %, 39.5 to 60mol %, 39.5 to 55 mol %, 39.5 to 52.5 mol %, 39.5 to 52 mol %, 39.5 to51 mol %, 39.5 to 50 mol %, 39.5 to 47.5 mol %, 39.5 to 45 mol %, 39.5to 44 mol %, 39.5 to 43.5 mol %, 39.5 to 43 mol %, 39.5 to 41.5 mol %,39.5 to 40 mol %, 40 to 60 mol %, 40 to 55 mol %, 40 to 52.5 mol %, 40to 52 mol %, 40 to 51 mol %, 40 to 50 mol %, 40 to 47.5 mol %, 40 to 45mol %, 40 to 44 mol %, 40 to 43.5 mol %, 40 to 43 mol %, 40 to 41.5 mol%, 41.5 to 60 mol %, 41.5 to 55 mol %, 41.5 to 52.5 mol %, 41.5 to 52mol %, 41.5 to 51 mol %, 41.5 to 50 mol %, 41.5 to 47.5 mol %, 41.5 to45 mol %, 41.5 to 44 mol %, 41.5 to 43.5 mol %, 41.5 to 43 mol %, 43 to60 mol %, 43 to 55 mol %, 43 to 52.5 mol %, 43 to 52 mol %, 43 to 51 mol%, 43 to 50 mol %, 43 to 47.5 mol %, 43 to 45 mol %, 43 to 44 mol %, 43to 43.5 mol %, 43.5 to 60 mol %, 43.5 to 55 mol %, 43.5 to 52.5 mol %,43.5 to 52 mol %, 43.5 to 51 mol %, 43.5 to 50 mol %, 43.5 to 47.5 mol%, 43.5 to 45 mol %, 43.5 to 44 mol %, 45 to 60 mol %, 45 to 55 mol %,45 to 52.5 mol %, 45 to 52 mol %, 45 to 51 mol %, 45 to 50 mol %, 45 to47.5 mol %, 47.5 to 60 mol %, 47.5 to 55 mol %, 47.5 to 52.5 mol %, 47.5to 52 mol %, 47.5 to 51 mol %, 47.5 to 50 mol %,%, 50 to 60 mol %, 50 to55 mol %, 50 to 52.5 mol %, 50 to 52 mol %, 50 to 52.5 mol % 50 to 51.5mol %, 51 to 60 mol %, 51 to 55 mol %, 51 to 52.5 mol %, or 51 to 52 mol%, %, 51 to 60 mol %, 51 to 55 mol %, 51 to 52.5 mol %, or 51 to 52 mol%.

According to one example, the sum of the lipid-PEG conjugate andcholesterol may be comprised in the lipid nanoparticle in an amount of30 to 70 mol %, 40 to 70 mol %, 40 to 60 mol %, 40 to 55 mol %, 40 to53.5 mol %, 40 to 50 mol %, 40 to 47.5 mol %, 40 to 45 mol %, 40 to 44.5mol %, 42 to 60 mol %, 42 to 55 mol %, 42 to 53.5 mol %, 42 to 50 mol %,42 to 47.5 mol %, 42 to 45 mol %, 42 to 44.5 mol %, 44 to 60 mol %, 44to 55 mol %, 44 to 53.5 mol %, 44 to 50 mol %, 44 to 47.5 mol %, 44 to45 mol %, 44 to 44.5 mol %, 44.5 to 60 mol %, 44.5 to 55 mol %, 44.5 to53.5 mol %, 44.5 to 50 mol %, 44.5 to 47.5 mol %, or 44.5 to 45 mol %.

According to one example, the ionizable lipid may be comprised in thelipid nanoparticle in an amount of 10 to 70 mol %, 10 to 60 mol %, 10 to55 mol %, 10 to 50 mol %, 10 to 45 mol %, 10 to 42.5 mol %, 10 to 40 mol%, 10 to 35 mol %, 10 to 30 mol %, 10 to 26.5 mol %, 10 to 25 mol %, 10to 20 mol %, 15 to 60 mol %, 15 to 55 mol %, 15 to 50 mol %, 15 to 45mol %, 15 to 42.5 mol %, 15 to 40 mol %, 15 to 35 mol %, 15 to 30 mol %,15 to 26.5 mol %, 15 to 25 mol %, 15 to 20 mol %, 20 to 60 mol %, 20 to55 mol %, 20 to 50 mol %, 20 to 45 mol %, 20 to 42.5 mol %, 20 to 40 mol%, 20 to 35 mol %, 20 to 30 mol %, 20 to 26.5 mol %, 20 to 25 mol %, 25to 60 mol %, 25 to 55 mol %, 25 to 50 mol %, 25 to 45 mol %, 25 to 42.5mol %, 25 to 40 mol %, 25 to 35 mol %, 25 to 30 mol %, 25 to 26.5 mol %,26.5 to 60 mol %, 26.5 to 55 mol %, 26.5 to 50 mol %, 26.5 to 45 mol %,26.5 to 42.5 mol %, 26.5 to 40 mol %, 26.5 to 35 mol %, 26.5 to 30 mol%, 30 to 60 mol %, 30 to 55 mol %, 30 to 50 mol %, 30 to 45 mol %, 30 to42.5 mol %, 30 to 40 mol %, 30 to 35 mol %, 35 to 60 mol %, 35 to 55 mol%, 35 to 50 mol %, 35 to 45 mol %, 35 to 42.5 mol %, 35 to 40 mol %, 40to 60 mol %, 40 to 55 mol %, 40 to 50 mol %, 40 to 45 mol %, 40 to 42.5mol %, 42.5 to 60 mol %, 42.5 to 55 mol %, 42.5 to 50 mol %, or 42.5 to45 mol %.

According to one example, the phospholipid may be comprised in the lipidnanoparticle in an amount of 1 to 50 mol %, 5 to 50 mol %, 5 to 40 mol%, 5 to 30 mol %, 5 to 25 mol %, 5 to 20 mol %, 5 to 15 mol %, 5 to 13mol %, 5 to 10 mol %, 10 to 30 mol %, 10 to 25 mol %, 10 to 20 mol %, 10to 15 mol %, 10 to 13 mol %, 15 to 30 mol %, 15 to 25 mol %, 15 to 20mol %, 20 to 30 mol %, or 20 to 25 mol %.

Herein, “mol % (mol %, mole percentage)” is expressed as a percentage bydividing the number of moles of a specific component by the sum of molesof all components and then multiplying by 100, and expressed as aformula, for example, it may be as Equation 1 below.

$\begin{matrix}\begin{array}{l}\text{mol \%=} \\{\text{(moles}\,\text{of}\,\text{a}\,\text{specific}\,\text{component)/(sum}\,\text{of}\,\text{moles}\,\text{of}\,\text{all}\,\text{components)} \times 100}\end{array} & \text{­­­(Equation 1)}\end{matrix}$

The lipid nanoparticle may comprise the ionizablelipid:phospholipid:cholesterol:lipid-PEG conjugate at a molar ratio of20 to 50:10 to 30:20 to 60:0.1 to 10, at a molar ratio of 20 to 50:10 to30:20 to 60:0.25 to 10, at a molar ratio of 20 to 50:10 to 30:30 to60:0.25 to 10, at a molar ratio of 20 to 50:10 to 30:30 to 60:0.1 to 5,at a molar ratio of 20 to 50: 10 to 30:30 to 60:0.5 to 5, at a molarratio of 25 to 45:10 to 25:40 to 50:0.5 to 3, at a molar ratio of 25 to45:10 to 20:40 to 55:0.5 to 3, at a molar ratio of 25 to 45:10 to 20:40to 55:1.0 to 1.5, at a molar ratio of 40 to 45:10 to 15:40 to 45:0.5 to3.0, at a molar ratio of 40 to 45: 10 to 15:40 to 45:0.5 to 3, at amolar ratio of 40 to 45:10 to 15:40 to 45:1 to 1.5, at a molar ratio of25 to 30:17 to 22; 50 to 55:0.5 to 3.0, at a molar ratio of 25 to 30:17to 22; 50 to 55:1.0 to 2.5, or at a molar ratio of 25 to 30:17 to 22; 50to 55:1.5 to 2.5. According to one example, while maintaining the sum ofthe moles of the lipid-PEG conjugate and cholesterol constant, among thecomponents comprised in the lipid nanoparticle, the moles of cholesterolare decreased as much as the number of moles of the lipid-PEG conjugateis increased, and thereby the molar ratio of the components can bemaintained.

Herein, the molar ratio means a ratio of moles, and “part of weight”mean a weight ratio in which each component is comprised.

In one example, the lipid nanoparticle may comprise the ionizable lipidof 20 to 50 parts by weight, phospholipid of 10 to 30 parts by weight,cholesterol of 20 to 60 parts by weight (or 20 to 60 parts by weight),and lipid-PEG conjugate of 0.1 to 10 parts by weight (or 0.25 to 10parts by weight, 0.5 to 5 parts by weight). The lipid nanoparticle maycomprise the ionizable lipid of 20 to 50% by weight, phospholipid of 10to 30% by weight, cholesterol of 20 to 60% by weight (or 30 to 60% byweight), and lipid-PEG conjugate of 0.1 to 10% by weight (or 0.25 to 10%by weight, 0.5 to 5% by weight) based on the total nanoparticle weight.In other example, the lipid nanoparticle may comprise the ionizablelipid of 25 to 50% by weight, phospholipid of 10 to 20% by weight,cholesterol of 35 to 55% by weight, and lipid-PEG conjugate of 0.1 to10% by weight (or 0.25 to 10% by weight, 0.5 to 5% by weight), based onthe total nanoparticle weight.

The lipid nanoparticle comprising the ionizable lipid, cholesterol,phospholipid, and/or lipid-PEG conjugate in the above range (molarratio, part by weight, and/or % by weight) may have excellent (i)stability of the lipid nanoparticle, (ii) encapsulation efficiency ofthe drug, and/or (iii) drug delivery efficiency targeting liver tissueand/or cells (for example, hepatocytes and/or LSEC), than the lipidnanoparticle comprising the ionizable lipid, cholesterol, phospholipidand/or lipid-PEG conjugate outside the above range.

The lipid nanoparticle according to one example may have an averagediameter of 20 nm to 200 nm, 20 to 180 nm, 20 nm to 170 nm, 20 nm to 150nm, 20 nm to 120 nm, 20 nm to 100 nm, 20 nm to 90 nm, 30 nm to 200 nm,30 to 180 nm, 30 nm to 170 nm, 30 nm to 150 nm, 30 nm to 120 nm, 30 nmto 100 nm, 30 nm to 90 nm, 40 nm to 200 nm, 40 to 180 nm, 40 nm to 170nm, 40 nm to 150 nm, 40 nm to 120 nm, 40 nm to 100 nm, 40 nm to 90 nm,50 nm to 200 nm, 50 to 180 nm, 50 nm to 170 nm, 50 nm to 150 nm, 50 nmto 120 nm, 50 nm to 100 nm, 50 nm to 90 nm, 60 nm to 200 nm, 60 to 180nm, 60 nm to 170 nm, 60 nm to 150 nm, 60 nm to 120 nm, 60 nm to 100 nm,60 nm to 90 nm, 70 nm to 200 nm, 70 to 180 nm, 70 nm to 170 nm, 70 nm to150 nm, 70 nm to 120 nm, 70 nm to 100 nm, 70 nm to 90 nm, 80 nm to 200nm, 80 to 180 nm, 80 nm to 170 nm, 80 nm to 150 nm, 80 nm to 120 nm, 80nm to 100 nm, 80 nm to 90 nm, 90 nm to 200 nm, 90 to 180 nm, 90 nm to170 nm, 90 nm to 150 nm, 90 nm to 120 nm, or 90 nm to 100 nm, for easyintroduction into liver tissue, hepatocytes and/or LSEC (liversinusoidal endothelial cells). When the size of the lipid nanoparticleis smaller than the above range, it is difficult to maintain stabilityas the surface area of the lipid nanoparticle is excessively increased,and thus delivery to the target tissue and/or drug effect may bereduced.

The liver tissue, hepatocytes and/or LSEC targeting effect (drugdelivery effect) of the lipid nanoparticle according to one example maybe dependent on the size of the lipid nanoparticle. For example, in caseof the lipid nanoparticle having a diameter of 40 to 120 nm, 30 to 100nm, 35 to 95 nm, 40 to 90 nm, 45 to 90 nm, 50 to 90 nm, 55 to 85 nm, 60to 80 nm, 70 to 90 nm, 70 to 80 nm, 50 to 70 nm, or 60 to 70 nm, thetargeting effect to hepatocytes may be excellent (than the nanoparticlehaving a diameter outside the above range). As another example, in caseof the lipid nanoparticle having a diameter of 20 to 200 nm, 20 to 180nm, 40 to 180 nm, 40 to 170 nm, 50 to 160 nm, 70 to 180 nm, 70 to 170nm, 75 to 170 nm, 75 to 165 nm, 70 to 150 nm, 70 to 130 nm, 75 to 130nm, 80 to 120 nm, 85 to 120 nm, about 90 to 120 nm, 90 to 110 nm, 90 to100 nm, 80 to 110 nm, 80 to 100 nm, 85 to 95 nm, about 90 nm, or 90 nm,the targeting effect to LSEC may be excellent (than the nanoparticlehaving a diameter outside the above range).

The lipid nanoparticle may specifically target liver tissue. The lipidnanoparticle according to one example may imitate metabolic behaviors ofnatural lipoproteins very similarly, and may be usefully applied for thelipid metabolism process by the liver and therapeutic mechanism throughthis.

The lipid nanoparticle may target hepatocytes. When the content of thelipid-PEG content comprised in the lipid nanoparticle is 0.1 to 15 mol%, 0.25 to 15 mol %, 0.5 to 15 mol %, 1 to 15 mol %, 1.5 to 15 mol %, 2to 15 mol %, 2.5 to 15 mol %, 0.1 to 12.5 mol %, 0.25 to 12.5 mol %, 0.5to 12.5 mol %, 1 to 12.5 mol %, 1.5 to 12.5 mol %, 2 to 12.5 mol %, 2.5to 12.5 mol %, 0.1 to 10 mol %, 0.25 to 10 mol %, 0.5 to 10 mol %, 1 to10 mol %, 1.5 to 10 mol %, 2 to 10 mol %, 2.5 to 10 mol %, 0.1 to 7.5mol %, 0.25 to 7.5 mol %, 0.5 to 7.5 mol %, 1 to 7.5 mol %, 1.5 to 7.5mol %, 2 to 7.5 mol %, 2.5 to 7.5 mol %, 0.1 to 5 mol %, 0.25 to 5 mol%, 0.5 to 5 mol %, 1 to 5 mol %, 1.5 to 5 mol %, 2 to 5 mol %, 2.5 to 5mol %, 0.1 to 3 mol %, 0.25 to 3 mol %, 0.5 to 3 mol %, 1 to 3 mol %,1.5 to 3 mol %, 2 to 3 mol %, 2.5 to 3 mol %, 0.1 to 2.5 mol %, 0.25 to2.5 mol %, 0.5 to 2.5 mol %, 1 to 2.5 mol %, 1.5 to 2.5 mol %, 2 to 2.5mol %, 0.1 to 4.5 mol %, 0.25 to 4.5 mol %, 0.5 to 4.5 mol %, 1 to 4.5mol %, 1.5 to 4.5 mol %, 2 to 4.5 mol %, 2.5 to 4.5 mol %, 0.1 to 4 mol%, 0.25 to 4 mol %, 0.5 to 4 mol %, 1 to 4 mol %, 1.5 to 4 mol %, 2 to 4mol %, 2.5 to 4 mol %, 0.1 to 3.5 mol %, 0.25 to 3.5 mol %, 0.5 to 3.5mol %, 1 to 3.5 mol %, 1.5 to 3.5 mol %, 2 to 3.5 mol %, 2.5 to 3.5 mol%, 0.1 to 2.0 mol %, 0.1 to 1.5 mol %, 0.1 to 1.0 mol %, 0.5 to 2.0 mol%, 0.5 to 1.5 mol %, 0.5 to 1.0 mol %, 1.0 to 2.0 mol %, 1.0 to 1.5 mol%, 1.5 to 2.0 mol %, 1.0 mol %, or 1.5 mol %, the drug deliveryefficiency (hepatocyte targeting efficiency) to hepatocytes of the lipidnanoparticle may be excellent.

The lipid nanoparticle may target an LSEC (liver sinusoidal endothelialcell). When the content of the lipid-PEG content comprised in the lipidnanoparticle is 0.1 to 15 mol %, 0.25 to 15 mol %, 0.5 to 15 mol %, 1 to15 mol %, 1.5 to 15 mol %, 2 to 15 mol %, 2.5 to 15 mol %, 0.1 to 12.5mol %, 0.25 to 12.5 mol %, 0.5 to 12.5 mol %, 1 to 12.5 mol %, 1.5 to12.5 mol %, 2 to 12.5 mol %, 2.5 to 12.5 mol %, 0.1 to 10 mol %, 0.25 to10 mol %, 0.5 to 10 mol %, 1 to 10 mol %, 1.5 to 10 mol %, 2 to 10 mol%, 2.5 to 10 mol %, 0.1 to 7.5 mol %, 0.25 to 7.5 mol %, 0.5 to 7.5 mol%, 1 to 7.5 mol %, 1.5 to 7.5 mol %, 2 to 7.5 mol %, 2.5 to 7.5 mol %,0.1 to 5 mol %, 0.25 to 5 mol %, 0.5 to 5 mol %, 1 to 5 mol %, 1.5 to 5mol %, 2 to 5 mol %, 2.5 to 5 mol %, 0.1 to 3 mol %, 0.25 to 3 mol %,0.5 to 3 mol %, 1 to 3 mol %, 1.5 to 3 mol %, 2 to 3 mol %, 2.5 to 3 mol%, 0.1 to 2.5 mol %, 0.25 to 2.5 mol %, 0.5 to 2.5 mol %, 1 to 2.5 mol%, 1.5 to 2.5 mol %, 2 to 2.5 mol %, 0.1 to 4.5 mol %, 0.25 to 4.5 mol%, 0.5 to 4.5 mol %, 1 to 4.5 mol %, 1.5 to 4.5 mol %, 2 to 4.5 mol %,2.5 to 4.5 mol %, 0.1 to 4 mol %, 0.25 to 4 mol %, 0.5 to 4 mol %, 1 to4 mol %, 1.5 to 4 mol %, 2 to 4 mol %, 2.5 to 4 mol %, 0.1 to 3.5 mol %,0.25 to 3.5 mol %, 0.5 to 3.5 mol %, 1 to 3.5 mol %, 1.5 to 3.5 mol %, 2to 3.5 mol %, 2.5 to 3.5 mol %, 0.1 to 2.0 mol %, 0.1 to 1.5 mol %, 0.1to 1.0 mol %, 0.5 to 2.0 mol %, 0.5 to 1.5 mol %, 0.5 to 1.0 mol %, 1.0to 2.0 mol %, 1.0 to 1.5 mol %, 1.5 to 2.0 mol %, 1.0 mol %, or 1.5 mol%, the drug delivery efficiency (LSEC targeting efficiency) to LSEC ofthe lipid nanoparticle may be excellent.

Herein, “targeting” and “localization” to the liver tissue, hepatocytesand/or LSEC of the lipid nanoparticle may be internalization in thetissue or cells, and may mean internalization inside the nucleus as itcan penetrate the nuclear membrane.

As other aspect, a composition for drug delivery comprising (i) thelipid nanoparticle; and (ii) an anionic drug, nucleic acid orcombination thereof (combination of the anionic drug and nucleic acid)is provided. The drug may be an anionic drug, nucleic acid orcombination thereof (anionic drug and nucleic acid).

The composition for drug delivery may be what a bioactive substance suchas an anionic drug and/or nucleic acid, and the like may be encapsulatedinside the lipid nanoparticle, and the bioactive substance such as ananionic drug and/or nucleic acid may be encapsulated with stable andhigh efficiency and thereby, it may show an excellent therapeutic effectby the composition for delivery. In addition, there is an advantage ofvariously controlling the kinds of the drug to be encapsulated in thelipid nanoparticle according to the purpose of treatment.

The lipid nanoparticle may have an anionic drug and/or nucleic acidencapsulated inside (the lipid nanoparticle). For the lipid nanoparticlein which an anionic drug and/or nucleic acid is encapsulated (inside thelipid nanoparticle) is the same as for the lipid nanoparticle describedabove.

In one example, the weight ratio of the ionizable lipid and drug(anionic drug, nucleic acid or combination thereof) comprised in thelipid nanoparticle may be 1 to 20:1, 1 to 15:1, 1 to 10:1, 5 to 20:1, 5to 15:1, 5 to 10:1, 7.5 to 20:1, 7.5 to 15:1, or 7.5 to 10:1.

In one example, when the (i) ionizable lipid; and (2) drug (anionicdrug, nucleic acid or combination thereof) are comprised at a weightratio in the above range, the encapsulation efficiency of the drug(anionic drug, nucleic acid or combination thereof) inside the lipidnanoparticle and/or drug delivery efficiency may be higher than thelipid nanoparticle comprising the (1) ionizable lipid; and (2) anionicdrug, nucleic acid or combination thereof at a weight ratio outside theabove range.

The lipid nanoparticle in which the anionic drug and/or nucleic acid isencapsulated may have an average diameter of 20 nm to 200 nm, 20 to 180nm, 20 nm to 170 nm, 20 nm to 150 nm, 20 nm to 120 nm, 20 nm to 100 nm,20 nm to 90 nm, 30 nm to 200 nm, 30 to 180 nm, 30 nm to 170 nm, 30 nm to150 nm, 30 nm to 120 nm, 30 nm to 100 nm, 30 nm to 90 nm, 40 nm to 200nm, 40 to 180 nm, 40 nm to 170 nm, 40 nm to 150 nm, 40 nm to 120 nm, 40nm to 100 nm, 40 nm to 90 nm, 50 nm to 200 nm, 50 to 180 nm, 50 nm to170 nm, 50 nm to 150 nm, 50 nm to 120 nm, 50 nm to 100 nm, 50 nm to 90nm, 60 nm to 200 nm, 60 to 180 nm, 60 nm to 170 nm, 60 nm to 150 nm, 60nm to 120 nm, 60 nm to 100 nm, 60 nm to 90 nm, 70 nm to 200 nm, 70 to180 nm, 70 nm to 170 nm, 70 nm to 150 nm, 70 nm to 120 nm, 70 nm to 100nm, 70 nm to 90 nm, 80 nm to 200 nm, 80 to 180 nm, 80 nm to 170 nm, 80nm to 150 nm, 80 nm to 120 nm, 80 nm to 100 nm, 80 nm to 90 nm, 90 nm to200 nm, 90 to 180 nm, 90 nm to 170 nm, 90 nm to 150 nm, 90 nm to 120 nm,or 90 nm to 100 nm, so that the introduction into the liver tissue,hepatocytes and/or LSEC (liver sinusoidal endothelial cells) is easy.

When the size of the lipid nanoparticle is smaller than the lower limitof the above range, (i) during systemic circulation of the lipidnanoparticle, binding of apolipoproteins (for example, ApoE (e.g.ApoE3)) present in blood is reduced, and therefore, the number of thelipid nanoparticles entering the cells may be reduced and/or (ii) thesurface area of the lipid nanoparticle is excessively increased andtherefore it is difficult to maintain the stability, and thus the drugdelivery efficiency to target tissue (or target cell) and/or therapeuticeffect of the drug which the lipid nanoparticle carries out may bereduced.

The lipid nanoparticle having a diameter in the above range hasexcellent drug delivery efficiency to a target organ and/or cell thanthe lipid nanoparticle having a diameter over the upper limit of theabove range.

In one example, the composition for drug delivery comprising (1) thelipid nanoparticle; and (2) anionic drug, nucleic acid or combinationthereof may be a composition for drug delivery to hepatocytes.

According to one example, the diameter of the lipid nanoparticlecomprised in the composition for drug (anionic drug, nucleic acid orcombination thereof) delivery to hepatocytes may be 40 to 120 nm, 30 to100 nm, 35 to 95 nm, 40 to 90 nm, 45 to 90 nm, 50 to 90 nm, 55 to 85 nm,60 to 80 nm, 70 to 90 nm, 70 to 80 nm, 50 to 70 nm, or 60 to 70 nm.During the drug delivery to hepatocytes, the diameter of the fenestraeleading from the sinusoidal lumen to the hepatocytes is about 140 nm inmammals and about 100 nm in humans, so the composition for drug deliveryhaving a diameter in the above range may have excellent drug deliveryefficiency to hepatocytes than the lipid nanoparticle having thediameter outside the above range.

According to one example, the lipid nanoparticle comprised in thecomposition for drug delivery to hepatocytes may comprise the lipid-PEGconjugate in an amount of 0.1 to 15 mol %, 0.25 to 15 mol %, 0.5 to 15mol %, 1 to 15 mol %, 1.5 to 15 mol %, 2 to 15 mol %, 2.5 to 15 mol %,0.1 to 12.5 mol %, 0.25 to 12.5 mol %, 0.5 to 12.5 mol %, 1 to 12.5 mol%, 1.5 to 12.5 mol %, 2 to 12.5 mol %, 2.5 to 12.5 mol %, 0.1 to 10 mol%, 0.25 to 10 mol %, 0.5 to 10 mol %, 1 to 10 mol %, 1.5 to 10 mol %, 2to 10 mol %, 2.5 to 10 mol %, 0.1 to 7.5 mol %, 0.25 to 7.5 mol %, 0.5to 7.5 mol %, 1 to 7.5 mol %, 1.5 to 7.5 mol %, 2 to 7.5 mol %, 2.5 to7.5 mol %, 0.1 to 5 mol %, 0.25 to 5 mol %, 0.5 to 5 mol %, 1 to 5 mol%, 1.5 to 5 mol %, 2 to 5 mol %, 2.5 to 5 mol %, 0.1 to 3 mol %, 0.25 to3 mol %, 0.5 to 3 mol %, 1 to 3 mol %, 1.5 to 3 mol %, 2 to 3 mol %, 2.5to 3 mol %, 0.1 to 2.5 mol %, 0.25 to 2.5 mol %, 0.5 to 2.5 mol %, 1 to2.5 mol %, 1.5 to 2.5 mol %, 2 to 2.5 mol %, 0.1 to 4.5 mol %, 0.25 to4.5 mol %, 0.5 to 4.5 mol %, 1 to 4.5 mol %, 1.5 to 4.5 mol %, 2 to 4.5mol %, 2.5 to 4.5 mol %, 0.1 to 4 mol %, 0.25 to 4 mol %, 0.5 to 4 mol%, 1 to 4 mol %, 1.5 to 4 mol %, 2 to 4 mol %, 2.5 to 4 mol %, 0.1 to3.5 mol %, 0.25 to 3.5 mol %, 0.5 to 3.5 mol %, 1 to 3.5 mol %, 1.5 to3.5 mol %, 2 to 3.5 mol %, 2.5 to 3.5 mol %, 0.1 to 2.0 mol %, 0.1 to1.5 mol %, 0.1 to 1.0 mol %, 0.5 to 2.0 mol %, 0.5 to 1.5 mol %, 0.5 to1.0 mol %, 1.0 to 2.0 mol %, 1.0 to 1.5 mol %, 1.5 to 2.0 mol %, 1.0 mol%, or 1.5 mol %, and the lipid nanoparticle comprising the lipid-PEGconjugate in the above range may have excellent drug delivery efficiencyspecific to hepatocytes (or targeting hepatocytes).

According to one example, the lipid nanoparticle comprised in thecomposition for drug delivery to hepatocytes may comprise the ionizablelipid : phospholipid : cholesterol: lipid-PEG conjugate in the rangedescribed above or at a molar ratio of 20 to 50:10 to 30:30 to 60:0.5 to5, at a molar ratio of 25 to 45:10 to 25:40 to 50:0.5 to 3, at a molarratio of 25 to 45: 10 to 20:40 to 55:0.5 to 3, or at a molar ratio of 25to 45:10 to 20:40 to 55:1.0 to 1.5. The lipid nanoparticle comprisingcomponents at the molar ratio in the above range may have excellent drugdelivery efficiency specific to hepatocytes (or targeting hepatocytes).

In one example, the composition for drug delivery comprising (1) thelipid nanoparticle; and (2) anionic drug, nucleic acid or combinationthereof may be a composition for drug delivery into LSEC.

When the diameter of the lipid nanoparticle comprised in the compositionfor drug delivery into LSEC is similar to or slightly smaller than thediameter of the fenestrae, the drug delivery effect into LSEC may beexcellent. In one example, the diameter of the lipid nanoparticlecomprised in the composition for drug delivery into LSEC may be 20 to200 nm, 20 to 180 nm, 40 to 180 nm, 40 to 170 nm, 50 to 160 nm, 70 to180 nm, 70 to 170 nm, 75 to 170 nm, 75 to 165 nm, 70 to 150 nm, 70 to130 nm, 75 to 130 nm, 80 to 120 nm, 85 to 120 nm, about 90 to 120 nm, 90to 110 nm, 80 to 110 nm, 80 to 100 nm, 85 to 95 nm, about 90 nm, or 90nm.

According to one example, the lipid nanoparticle comprised in thecomposition for drug delivery into LSEC may comprise the lipid-PEGconjugate in an amount of 0.1 to 15 mol %, 0.25 to 15 mol %, 0.5 to 15mol %, 1 to 15 mol %, 1.5 to 15 mol %, 2 to 15 mol %, 2.5 to 15 mol %,0.1 to 12.5 mol %, 0.25 to 12.5 mol %, 0.5 to 12.5 mol %, 1 to 12.5 mol%, 1.5 to 12.5 mol %, 2 to 12.5 mol %, 2.5 to 12.5 mol %, 0.1 to 10 mol%, 0.25 to 10 mol %, 0.5 to 10 mol %, 1 to 10 mol %, 1.5 to 10 mol %, 2to 10 mol %, 2.5 to 10 mol %, 0.1 to 7.5 mol %, 0.25 to 7.5 mol %, 0.5to 7.5 mol %, 1 to 7.5 mol %, 1.5 to 7.5 mol %, 2 to 7.5 mol %, 2.5 to7.5 mol %, 0.1 to 5 mol %, 0.25 to 5 mol %, 0.5 to 5 mol %, 1 to 5 mol%, 1.5 to 5 mol %, 2 to 5 mol %, 2.5 to 5 mol %, 0.1 to 3 mol %, 0.25 to3 mol %, 0.5 to 3 mol %, 1 to 3 mol %, 1.5 to 3 mol %, 2 to 3 mol %, 2.5to 3 mol %, 0.1 to 2.5 mol %, 0.25 to 2.5 mol %, 0.5 to 2.5 mol %, 1 to2.5 mol %, 1.5 to 2.5 mol %, 2 to 2.5 mol %, 0.1 to 4.5 mol %, 0.25 to4.5 mol %, 0.5 to 4.5 mol %, 1 to 4.5 mol %, 1.5 to 4.5 mol %, 2 to 4.5mol %, 2.5 to 4.5 mol %, 0.1 to 4 mol %, 0.25 to 4 mol %, 0.5 to 4 mol%, 1 to 4 mol %, 1.5 to 4 mol %, 2 to 4 mol %, 2.5 to 4 mol %, 0.1 to3.5 mol %, 0.25 to 3.5 mol %, 0.5 to 3.5 mol %, 1 to 3.5 mol %, 1.5 to3.5 mol %, 2 to 3.5 mol %, 2.5 to 3.5 mol %, 0.1 to 2.0 mol %, 0.1 to1.5 mol %, 0.1 to 1.0 mol %, 0.5 to 2.0 mol %, 0.5 to 1.5 mol %, 0.5 to1.0 mol %, 1.0 to 2.0 mol %, 1.0 to 1.5 mol %, 1.5 to 2.0 mol %, 1.0 mol%, or 1.5 mol %, and the lipid nanoparticle comprising the lipid-PEGconjugate in the above range may have excellent drug delivery efficiencyspecific to LSEC (or targeting LSEC).

According to one example, the lipid nanoparticle comprised in thecomposition for drug delivery into LSEC may comprise the ionizable lipid: phospholipid : cholesterol: lipid-PEG conjugate in the range describedabove or at a molar ratio of 20 to 50:10 to 30:30 to 60:0.5 to 5, at amolar ratio of 25 to 45:10 to 25:40 to 50:0.5 to 3, at a molar ratio of25 to 45: 10 to 20:40 to 55:0.5 to 3, or at a molar ratio of 25 to 45:10to 20:40 to 55:1.0 to 1.5. The lipid nanoparticle comprising componentsat a molar ratio in the above range may have excellent drug deliveryefficiency specific to LSEC (or targeting LSEC).

The lipid nanoparticle according to one example may delivery atherapeutic agent highly efficiently into liver tissue, hepatocytesand/or LSEC through liver tissue-specific properties, and may beusefully utilized for a drug for treating liver diseases, a deliverymethod of a gene for treatment, and the like, and a therapeutic agentmediated by liver, which mediate this highly efficient liver tissue,hepatocytes and/or LSEC-specific lipid nanoparticle. In addition, thelipid nanoparticle according to one example forms a stable complex witha gene drug such as nucleic acid, and the like, and shows lowcytotoxicity and effective cell absorption, and thus it is effective todeliver the gene drug such as nucleic acid.

The lipid nanoparticle is same as described above.

The lipid nanoparticle according to one example exhibits a positivecharge under the acidic pH condition by showing a pKa of 5 to 8, 5.5 to7.5, 6 to 7, or 6.5 to 7, and may encapsulate a drug with highefficiency by easily forming a complex with a drug through electrostaticinteraction with a therapeutic agent such as a nucleic acid and anionicdrug (for example, protein) showing a negative charge, and it may beusefully used as a composition for intracellular or in vivo drugdelivery of a drug (for example, nucleic acid).

Herein, “encapsulation” refers to encapsulating a delivery substance forsurrounding and embedding it in vivo efficiently, and the drugencapsulation efficiency (encapsulation efficiency) mean the content ofthe drug encapsulated in the lipid nanoparticle for the total drugcontent used for preparation.

The encapsulation efficiency of the anionic or nucleic acid drug of thecomposition for delivery may be 70% or more, 75% or more, 80% or more,85% or more, 90% or more, 91% or more, 92% or more, 94% or more, over80% to 99% or less, over 80% to 97% or less, over 80% to 95% or less,85% or more to 95% or less, 87% or more to 95% or less, 90% or more to95% or less, 91% or more to 95% or less, 91% or more to 94% or less,over 91% to 95% or less, 92% or more to 99% or less, 92% or more to 97%or less, or 92% or more to 95% or less.

According to one example, the encapsulation efficiency may be calculatedby commonly used methods, and for example, the drug encapsulationefficiency may be calculated by the following Equation 2, by treatingTriton-X to the lipid nanoparticle according to one example andmeasuring the fluorescence intensity of the Triton-X-treated andTriton-X-untreated lipid nanoparticles in a specific wavelengthbandwidth (for example, excitation: 480 ^(\~)490 nm, emission: 520^(\~)530 nm).

$\begin{matrix}\begin{array}{l}{\text{Drug encapsulation efficiency (\%)=(Fluorescence intensity (fluorescence) of the Triton-}} \\\text{X-treated lipid nanoparticle-Fluorescence intensity (fluorescence) of the Triton-X-untreated} \\\text{lipid nanoparticle)/(Fluorescence intensity (fluorescence) of the Triton-X-treated lipid} \\{\text{nanoparticle)} \times 100}\end{array} & \text{­­­(Equation 2)}\end{matrix}$

The composition for drug delivery according to one example may compriseCas9 mRNA with high encapsulation efficiency. The previously knowncomposition for delivering Cas9 mRNA has a limitation in using it as acomposition for delivering Cas9 mRNA, as it comprises Cas9 mRNA at a lowratio. On the other hand, the lipid nanoparticle according to oneexample may comprise Cas9 mRNA with high encapsulation efficiency,specifically, encapsulation efficiency of 70% or more, and thus it maybe usefully utilized for gene editing therapy.

The anionic drug may be an anionic biopolymer-drug conjugate such asvarious kinds of peptides, protein drugs, protein-nucleic acidstructures or hyaluronic acid-peptide conjugates, hyaluronicacid-protein conjugates, which have an anion, and the like. Thenon-restrictive examples of the protein drugs may be apoptosis-inducingfactors (e.g., cytochrome C, caspase 3/7/8/9, etc.) and including geneediting proteins such as Cas 9, cpf1 which are gene editing scissors,and various intracellular proteins (e.g., transcription factors), andthe like.

The nucleic acid may be one or more kinds selected from the groupconsisting of small interfering RNA (siRNA), ribosome ribonucleic acid(rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA),complementary deoxyribonucleic acid (cDNA), aptamer, messengerribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisenseoligonucleotide, shRNA, miRNA, ribozyme, PNA, DNAzyme and sgRNA for geneediting, and the like, but not limited thereto.

Herein, the term “siRNA” refers to double stranded RNA (duplex RNA)which can induce RNAi (RNA interference) through cleavage of specificmRNA, or single stranded RNA that has a double stranded form inside thesingle stranded RNA. It consists of a sense RNA strand having thesequence homologous to mRNA of a target gene and an antisense RNA strandhaving the sequence complementary thereto. As siRNA can inhibitexpression of a target gene, it is provided by an effective geneknock-down method or method of gene therapy. Binding between doublestrands is carried out through hydrogen bonding between nucleotides, andnot all nucleotides within the double strand must be complementary andcompletely bound.

The length of siRNA may be about 15 to 60, specifically about 15 to 50,about 15 to 40, about 15 to 30, about 15 to 25, about 16 to 25, about 19to 25, about 20 to 25, or about 20 to 23 nucleotides. The siRNA lengthmeans the number of nucleotides on one side of the double stranded RNA,that is, the number of base pairs, and in case of single stranded RNA,means the length of the double strand inside the single stranded RNA. Inaddition, siRNA may be composed of nucleotides introduced with variousfunctional groups for the purpose of increasing blood stability orweakening an immune response, and the like.

Herein, the term “antisense oligonucleotide” may be modified at aposition of one or more bases, sugars or backbones to enhance theefficacy (De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55,1995). The oligonucleotide backbone may be modified by phosphorothioate,phosphotriester, methyl phosphonate, short-chain alkyl, cycloalkyl,short-chain heteroatomic, heterocyclic intersaccharide binding, and thelike. In addition, the antisense oligonucleotide may comprise one ormore substituted sugar moieties. The antisense oligonucleotide maycomprise a modified base. The modified base includes hypoxanthine,6-methyladenine, 5-me pyrimidine (particularly, 5-methylcytosine),5-hydroxymethylcytosine (HMC), glycosyl HMC, gentiobiosyl HMC,2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil,5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine,N6(6-aminohexyl)adenine, 2,6-diaminopurine, and the like.

Herein, “single stranded deoxyribonucleic acid (ssDNA)” means a singlestranded oligonucleotide which binds to specific target DNA selectivelyand induces an antigene effect.

Herein, “aptamer” means an oligonucleotide (generally, 20 - 80 nt DNA orRNA) which binds to a specific target. Preferably, herein, “aptamer”means an oligonucleotide aptamer (e.g., DNA or RNA aptamer).

Herein, “mRNA” means synthetic mRNA (in vitro transcribed mRNA) capableof expressing a gene.

Herein, “shRNA” means single-stranded 50 to 70 nucleotides, and forms astem-loop (stemloop) structure in vivo. On both sides of the loop of 5to 10 nucleotides, complementarily, long RNA of 19 to 29 nucleotides isbase-paired to form a double-stranded stem.

Herein, “miRNA (microRNA)” means a single stranded RNA molecule whichcontrols gene expression and consists of full length 21 to 23nucleotides. miRNA is an oligonucleotide which is not expressed in acell, and has a short stem-loop structure. miRNA has full or partialhomology with one or two or more mRNA (messenger RNA) and suppressestarget gene expression through complementary binding to the mRNA.

Herein, “ribozyme” is a kind of RNA, and is RNA which recognizes anucleotide sequence of specific RNA and has the same function as enzymecutting it by itself. The ribozyme is a complementary nucleotidesequence of a target messenger RNA strand and consists of a region thatbinds with specificity and a region that cuts the target RNA.

Herein, “DNAzyme” is a single stranded DNA molecule having enzymeactivity, and DNAzyme consisting of 10 to 23 nucleotides (10-23 DNAzyme)cuts a RNA strand at a specific position under the physiologicallysimilar condition. The 10-23 DNAzyme cuts between any exposed purinesand pyrimidines without base pairing. The 10-23 DNAzyme consists of anactive site (catalytic domain) of enzyme consisting of 15 conservednucleotide sequences (for example, 5′-GGCTAGCTACAACGA-3′) and an RNAsubstrate binding domain consisting of 7″8 DNA nucleotide sequenceswhich recognize RNA substrates to the left and right of the activedomain of the enzyme afore-described.

Herein, “PNA (Peptide nucleic acid)” is a molecule having all propertiesof nucleic acids and proteins, and means a molecule capable ofcomplementarily binding to DNA or RNA. The PNA was first reported in1999 as similar DNA in which nucleobases are linked by peptide bonds(document [Nielsen P E, Egholm M, Berg R H, Buchardt O,“Sequence-selective recognition of DNA by strand displacement with athymine-substituted polyamide”, Science 1991, Vol. 254: pp 1497-1500]).The PNA is not found in the natural world and is artificiallysynthesized by a chemical method. The PNA causes a hybridizationreaction with a natural nucleic acid of a complementary nucleotidesequence to form a double strand. PNA/DNA double strands are more stablethan DNA/DNA double strands for the same length. As a backbone ofpeptides, N-(2-aminoethyl)glycine repeatedly linked by amide bonds ismost commonly used, and in this case, the backbone of the peptidenucleic acid is electrically neutral different from the backbone of thenatural nucleic acid. 4 nucleotides present in PNA have almost the samespatial size and distance between nucleotides as in case of the naturalnucleic acid. The PNA is not only chemically more stable than thenatural nucleic acid, but also biologically stable because it is notdegraded by nuclease or protease.

Herein, “sgRNA” is an oligonucleotide (generally, RNA molecule) bindingto a specific DNA target, and means a complex single RNA molecule ofcrispr RNA (crRNA) and tracer (tracrRNA). It is an RNA molecule which isused for recognizing a specific DNA sequence with Cas9 nuclease in theCRISPR system and enables selective gene cleavage, and approximately,comprises a 20-nt sequence capable of complementarily binding to DNA,and has a total length of 100 nt.

Herein, “gene editing protein” refers to Cas9, spCas9, cjCas9, casX,CasY and Cpf1, and the like, and refers to a protein that recognizes atarget DNA nucleotide sequence with sgRNA to cause DNA cleavage.

The target cell to which a drug and/or nucleic acid is delivered by thelipid nanoparticle according to one example may be a hepatocyte and/orLSEC in vivo or isolated in vivo. The composition for drug deliveryand/or complex of the drug (anionic drug, nucleic acid or combinationthereof) and lipid nanoparticle according to one example may target orspecifically target a hepatocyte and/or LSEC. Accordingly, the lipidnanoparticle or composition for drug or nucleic acid delivery comprisingthe lipid nanoparticle according to one example may be for treatment ofacute or chronic liver diseases such as hepatic fibrosis, livercirrhosis, hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C,etc.), and in addition, it may be utilized as a composition for deliveryof a therapeutic agent (drug) absorbed through liver.

Other aspect provides a pharmaceutical composition for preventing ortreating liver disease, comprising (1) the lipid nanoparticle; and (2)an anionic drug, nucleic acid or combination thereof.

The lipid nanoparticle comprised in the pharmaceutical composition forpreventing or treating liver disease is same as the lipid nanoparticlecomprised in the composition for drug delivery afore-mentioned.

The anionic drug and nucleic acid comprised in the pharmaceuticalcomposition for preventing or treating liver disease is same as theanionic drug and nucleic acid comprised in the composition for drugdelivery afore-mentioned.

The pharmaceutical composition for preventing or treating liver diseaseaccording to one example may comprise a lipid nanoparticle in which ananionic drug and/or nucleic acid is encapsulated.

The liver disease may be one or more kinds selected from the groupconsisting of ATTR amyloidosis, hypercholesterolemia, hepatitis B virusinfection, acute liver failure, cirrhosis, and liver fibrosis.

The anionic drug may have an effect for preventing or treating liverdisease.

The nucleic acid may have an effect for preventing or treating liverdisease, and for example, it may be siRNA and/or miRNA which can inhibitexpression such as (1) TTR (Transthyretin) ((e.g., human TTR (protein:GenBank Accession Nos. NP_000362.1; gene: GenBank Accession Nos.NM_000371.4, etc.), mouse TTR (protein: GenBank Accession No.;NP_038725.1; gene: GenBank Accession No. NM_013697.5, etc.)), (2) PCSK9(Proprotein convertase subtilisin/kexin type 9) ((e.g., human PCSK9(protein: GenBank Accession Nos. NP_777596.2; gene: GenBank AccessionNos. NM_174936.4, etc.), mouse PCSK9 (protein: GenBank Accession No.NP_705793.1; gene: GenBank Accession No. NM_153565.2, etc.)), (3) HBV(Hepatitis B Virus) (e.g., HBV genotype A (e.g., GenBank Accession No.:X02763, X51970, or AF090842); HBV genotype B (e.g., GenBank AccessionNo.: D00329, AF100309, or AB033554); HBV genotype C (e.g., GenBankAccession No.: X04615, M12906, AB014381, AB042285, AB042284, AB042283,AB042282, AB026815, AB026814, AB026813, AB026812, or AB026811); HBVgenotype D (e.g., GenBank Accession No.: X65259, M32138, or X85254); HBVgenotype E (e.g., GenBank Accession No.: X75657 or AB032431); HBVgenotype F (e.g., GenBank Accession No.: X69798, AB036910, or AF223965);HBV genotype G (e.g., GenBank Accession No.: AF160501, AB064310, orAF405706) HBV genotype H (e.g., AY090454, AY090457, or AY090460) (4) Bax(BCL2 associated X)) ((e.g., human Bax (protein: GenBank Accession Nos.NP_001278357.1, NP_001278358.1, NP_001278359.1, NP_001278360.1,NP_004315.1; gene: GenBank Accession Nos. NM_001291428.2,NM_001291429.2, NM_001291430.1, NM_001291431.2, NM_004324.4, etc.),mouse Bax (protein: GenBank Accession No. NP_031553.1; gene: GenBankAccession No. NM_007527.3, etc.), (5) VEGF (Vascular endothelial growthfactor) (e.g., VEGFA ((e.g., human VEGFA (protein: GenBank AccessionNos. NP_001020537.2, NP_001020538.2, NP 001020539.2, NP_001020540.2,NP_001020541.2; gene: GenBank Accession Nos. NM_003376.6,NM_001025366.3, NM_001025367.3, NM_001025368.3, NM_001025369.3, etc.),mouse VEGFA (protein: GenBank Accession No. NP_001020421.2,NP_001020428.2, NP_001103736.1, NP_001103737.1, NP_001103738.1; gene:GenBank Accession No. NM_001025250.3, NM_001025257.3, NM_001110266.1,NM_001110267.1, NM_001110268.1, etc.); VEGFB ((e.g., human VEGFB(protein: GenBank Accession Nos. NP_001230662.1, NP_003368.1; gene:GenBank Accession Nos. NM_003377.5, NM_001243733.2, etc.), mouse VEGFB(protein: GenBank Accession No. NP_001172093.1, NP_035827.1; gene:GenBank Accession No. NM_001185164.1, NM_011697.3, etc.); VEGFC (e.g.,human VEGFC (protein: GenBank Accession Nos. NP_005420.1; gene: GenBankAccession Nos. NM_005429.5, etc.), mouse VEGFC (protein: GenBankAccession No.; NP_033532.1; gene: GenBank Accession No. NM_009506.2,etc.)), and/or (6) PDGF (Platelet-derived growth factor) (e.g., PDGFA((e.g., human PDGFA (protein: GenBank Accession Nos. NP_002598.4,NP_148983.1; gene: GenBank Accession Nos. NM_002607.5, NM_033023.4,etc.), mouse PDGFA (protein: GenBank Accession No. NP_032834.1,NP_001350200.1; gene: GenBank Accession No. NM_008808.4, NM_001363271.1,etc.)); PDGFB ((e.g., human PDGFB (protein: GenBank Accession Nos.NP_002599.1, NP_148937.1; gene: GenBank Accession Nos. NM_033016.3,NM_002608.4, etc.), mouse PDGFB (protein: GenBank Accession No.NP_035187.2; gene: GenBank Accession No. NM_011057.4, etc.)); PDGFC((e.g., human PDGFC (protein: GenBank Accession Nos. NP_057289.1; gene:GenBank Accession Nos. NM_016205.3, etc.), mouse PDGFC (protein: GenBankAccession No. NP_064355.1, NP_001344675.1; gene: GenBank Accession No.NM_019971.3, NM_001357746.1, etc.)); PDGFD (e.g., human PDGFD (protein:GenBank Accession Nos. NP_079484.1, NP_149126.1; gene: GenBank AccessionNos. NM_033135.4, NM_025208.5, etc.), mouse PDGFD (protein: GenBankAccession No. NP_082200.1, NP_001344326.1, NP_001344327.1; gene: GenBankAccession No. NM_027924.3, NM_001357397.1, NM_001357398.1, etc.)).

The pharmaceutical composition may be administered by various routesincluding parenteral administration into mammals including humans, andparenteral administration may be applied intravenously, subcutaneously,intraperitoneally or locally, and the dosage varies depending on thecondition and body weight of the patient, degree of disease, drug form,administration route and time, but may be appropriately selected bythose skilled in the art.

When the pharmaceutical composition according to one example isformulated, it is prepared by using a diluent or excipient such as afiller, an extender, a binding agent, a wetting agent, a disintegrant, asurfactant, and the like used commonly.

The formulation for parenteral administration includes a sterilizedaqueous solution, a non-aqueous solvent, a suspended solvent, emulsion,a lyophilized formulation, a suppository, and the like.

As the non-aqueous solvent and suspended solvent, propylene glycol,polyethylene glycol, plant oil such as olive oil, injectable ester suchas ethyl oleate, and the like may be used. As a base compound of thesuppository, witepsol, macrogol, tween 61, cacao butter, laurin butter,glycerol, gelatin, and the like.

The pharmaceutical composition according to one example is administeredin a pharmaceutically effective dose. Herein, “pharmaceuticallyeffective dose” means an amount sufficient to treat disease at areasonable benefit/risk ratio applicable to medical treatment, and theeffective dose level may be determined depending on factors includingthe patient’s disease type, severity, drug activity, drug sensitivity,administration time, administration route and excretion rate, treatmentperiod and concomitant drugs, and other factors well known in themedical field. The pharmaceutical composition according to one examplemay be administered as an individual therapeutic agent or may beadministered in combination with other therapeutic agents, and may beadministered sequentially or simultaneously with conventionaltherapeutic agents, and may be singly or multiply administered. It isimportant to administer an amount capable of obtaining the maximumeffect with a minimum amount without side effects, in consideration ofall of the above factors, and this may be easily determined by thoseskilled in the art.

Specifically, the effective dose of the compound according to thepresent invention may vary depending on the patient’s age, gender andbody weight, and may be administered daily or every other day, oradministered by dividing into 1 to 3 times a day. However, it may beincreased or reduced depending on the administration route, severity ofobesity, gender, body weight, age, and the like, and therefore, theabove dose does not limit the scope of the present invention in any way.

In one specific example, the pharmaceutical composition may beadministered in a dose of 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 1 to 10mg/kg, or 1 to 5 mg/kg, based on the concentration of the drug (anionicdrug, nucleic acid or combination thereof) comprised in thepharmaceutical composition.

Other aspect provides a method for prevention or treatment of liverdisease comprising administering a composition (for example, thepharmaceutical composition for preventing or treating liver disease),comprising (1) the lipid nanoparticle; and (2) an anionic drug, nucleicacid or combination thereof. The lipid nanoparticle, anionic drug,nucleic acid, pharmaceutical composition and liver disease are same asdescribed above.

According to one example, the method for prevention or treatment ofliver disease may further comprise confirming (selecting) a patient inneed of prevention and/or treatment of the liver disease, beforeadministering the composition.

A subject to which the method for treatment is applied means a mammalincluding mice, livestock, and the like, including humans who have ormay have liver disease, but not limited thereto. The pharmaceuticalcomposition comprising the lipid nanoparticle according to one examplecan effectively deliver the anionic drug and/or nucleic acid to liver,and thus it may treat the subject effectively.

According to one example, a method for prevention or treatment of liverdisease, comprising administering a pharmaceutically effective dose ofcomposition (for example, the pharmaceutical composition for preventingor treating liver disease), comprising (1) the lipid nanoparticle; and(2) an anionic drug, nucleic acid or combination thereof to a patientmay be provided. The pharmaceutically effective dose is same asdescribed above, and a suitable total daily amount may be determined bytreatment within the scope of correct medical judgement, and may beadministered once or divided into several times. However, a specifictherapeutically effective dose for a particular patient will be applieddifferently depending on various factors including the specificcomposition, the patient’s age, body weight, general health condition,gender and diet, administration time, administration route and excretionrate of the composition, treatment period, and drugs used together orconcurrently with the specific composition, in addition to the type andextent of the response to be achieved, and whether or not other agent isused, if necessary, and similar factors well known in the pharmaceuticalfield.

Advantageous Effects

According to one example, the lipid nanoparticle is livertissue-specific, have excellent biocompatibility and can deliver a genetherapeutic agent with high efficiency, and thus it can be usefully usedin related technical fields such as lipid nanoparticle mediated genetherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a shows an exemplary structure of the lipid nanoparticleaccording to one example, and FIG. 1 b shows an image of observing thenanoparticle according to one example by Cryo-TEM.

FIG. 2 C10 LNP to 243-C10 LNP) and FIG. 3 (244-C 10 LNP to 246-C10 LNP)show the result of measuring the fluorescence intensity shown by eachlipid nanoparticle in a solution having a range of pH 4.1 to pH 9.6,

FIG. 4 a and FIG. 4 b are results showing the intracellular genedelivery efficiency of each nanoparticle. Specifically, FIG. 4 a showsthe luminescence intensity measured by transforming LNP encapsulatingmRNA (luc mRNA) encoding luciferase into HeLa cell and then dissolvingthe cell, and FIG. 4 b shows the luminescence intensity measured bytransforming LNP encapsulating luc mRNA to a hepatocyte and thendissolving the cell. In FIG. 4 b , +ApoE refers to a group treated byApoE3, and -ApoE refers to a group untreated by ApoE3.

FIG. 5 a shows in vivo drug delivery distribution in a mouse to which244-C10 LNP to 246-C10 LNP with Luc mRNA encapsulated is administered,and FIG. 5 b shows drug delivery distribution to each organ of the mouseremoved from the mouse in which 246-C10 LNP with Luc mRNA encapsulatedis administered.

FIG. 6 shows the drug delivery efficiency of the lipid nanoparticle andthe size of the nanoparticle, depending on the content of lipid-PEGcomprised in the lipid nanoparticle in the mouse in which 246-C10 LNPcomprising lipid-PEG in an amount of 1.0 to 2.5 mol % is administered.

FIG. 7 shows the result of confirming the hepatocyte targetingpossibility according to the concentration of siFVII administered asencapsulated in the lipid nanoparticle through the expression of FVII.

FIG. 8 shows the size of the lipid nanoparticle and PDI value of thelipid nanoparticle according to the content of the lipid-PEG comprisedin the lipid nanoparticle (left table), and shows the result ofconfirming the in vivo drug delivery efficiency to the hepatocytethrough the expression of FVII (right graph).

FIG. 9 shows the size of the lipid nanoparticle and PDI value of thelipid nanoparticle according to the content of the lipid-PEG comprisedin the lipid nanoparticle (left table), and shows the result ofconfirming the in vivo drug delivery efficiency to the LSEC through theexpression of FVIII (right graph).

FIG. 10 shows the result of measuring the intracellular siRNA deliveryefficiency of the lipid nanoparticle comprising ceramide-PEG orDSPE-PEG.

MODE FOR INVENTION

The present invention will be described in more detail by the followingexamples, but the scope is not intended to be limited by the followingexamples.

Hereinafter, the present invention will be described in more detail byexamples. These examples are only for describing the present inventionin more detail, and it will be apparent to those skilled in the art thatthe scope of the present invention is not limited by these examplesaccording to the gist of the present invention.

Example 1. Preparation of Ionizable Lipids Example 1-1. Preparation ofIonizable Lipids

Ionizable lipids were synthesized by reacting the amine-based compoundsof Table 1 below comprising a 6-membered heterocyclic tertiary amine and1,2-epoxydodecane (hereinafter, C10) (Sigma-Aldrich, USA) at a molarratio of 1:n (n=primary amine×2+secondary amine× 1).

TABLE 1 Name Chemical formula 241

242

243

244

245

246

Specifically, each of 241 to 246 amines of the Table 1 and epoxide (C10)were added at a molar ratio of l:n (n=primary amine×2+secondary amine×1)in a 5 ml vial with a magnetic bar and reacted in a stirrer at 750 rpm,90° C. for 3 days. Then, after purifying with WELUX fine silica column(Intertec, Korea), the molecular weight of each ionizable lipid producedby the reaction was calculated and they were stored at a concentrationof 100 mg/ml using ethanol. The ionizable lipid produced by using 241amine and C10 was named ‘241-C10’, and other ionizable lipids producedby using other kinds of amines were named ‘used amine name (241 to246)-C10’ in the same way.

Example 1-2. Confirmation of Produced Ionizable Lipids

In order to confirm the ionizable lipids produced in the Example 1-1, 1HNMR was performed. Specifically, the ionizable lipid (246-C10)synthesized in Example 1-1 of 5 ug was prepared by diluting inCDCl.sub.3 (sigma, USA) 0.5 ml to 100 mmole concentration. Then, 0.5 mleach was put into a tube for 400 MHz NMR and the top was sealed, andthen sealed with parafilm to obtain NMR spectra using Agilent 400 MHZFT-NMR (Agilent, USA), and the result shows that the signal representingeach functional group of 246-C10 was saturated.

In addition, in order to confirm the ionizable lipids (241-C10 to246-C10) prepared in Example 1-1, MS analysis was performed.Specifically, the ionizable lipids were diluted in ethanol at aconcentration of 0.5 ppm or less and MS analysis was performed. Theequipment used for the analysis was 6230 LC/MS of Agilent Technologies(Palo Alto, USA) and the Zorbax SB-C18 (100 mm×2.1 mm i.d., 3.5 µm) ofAgilent Technologies was used for the separation tube, and two solventsof distilled water (A) containing 0.1% formic acid and acetonitrile (B)were gradient eluted. The solvent gradient of the mobile phase wasmaintained for 4 minutes until the ratio of the organic solventacetonitrile (B) was initially increased from 30% to 80% for 2 minutesand then the ratio of the organic solvent was lowered to 30% again andstabilized. The flow rate of the mobile phase was 300 µl/min, and then,the injection volume of the analyzer was 2 µl The result of performingthe MS analysis was shown in Table 2 below. As shown in Table 2, itcould be confirmed that the measured m/z ratio and calculated m/z ratioof the ionizable lipids were almost identical.

TABLE 2 Chemical formula Calculated m/z ratio Observed m/z ratio 241-C10C₃₂H₆₆N₂O₂ 510.87864 511.5201 242-C10 C₃₁H₆₄N₂O₂ 496.85206 497.5043243-C10 C₃₁H₆₅N₃O₂ 511.8667 513.5186 244-C10 C₄₂H₈₇N₃O₃ 682.15848682.6821 245-C10 C₄₃H₈₉N₃O₃ 696.18506 696.7045 246-C10 C₅₈H₁₂₀N₄O₄937.5978 937.9383

From the result, it could be confirmed that the ionizable lipids werewell made in Example 1.1.

Example 2. Preparation of Lipid Nanoparticles Example 2-1. Preparationof Lipid Nanoparticles

The ionizable lipids (241-C10 to 246-C10) prepared in the Example 1-1,cholesterol (Cholesterol powder, BioReagent, suitable for cell culture,≥99%, sigma, Korea), phospholipid (DSPC) (Avanti, US), and a lipid-PEGconjugate (ceramide-PEG conjugate; C16 PEG2000 Ceramide, Avanti, US)were dissolved in ethanol at a molar ratio of 42.5:13:43:1.5.

The ethanol in which the ionizable lipids, cholesterol, phospholipid andlipid-PEG were dissolved and acetate buffer were mixed with a microfluidmixing device (Benchtop Nanoassemblr; PNI, Canada) at a flow rate of 12ml/min in a volume ratio of 1:3, thereby preparing lipid nanoparticles(LNPs).

Example 2-2. Preparation of Nucleic Acid-Encapsulated LipidNanoparticles

The ionizable lipids (241-C10 to 246-C10) prepared in the Example 1-1,cholesterol (Cholesterol powder, BioReagent, suitable for cell culture,≥99%, sigma, Korea), phospholipid (DSPC or DOPE) (18:0 PC (DSPC), 18:1(Δ9-Cis) PE (DOPE), Avanti, US), and a lipid-PEG conjugate (ceramide-PEGconjugate; C16 PEG2000 Ceramid, Avanti, US) were dissolved in ethanol.An RNA therapeutic agent, mRNA (luciferase mRNA; SEQ ID NO: 1) 30 ug wasdiluted in sodium citrate 0.75 ml, or siRNA (siFVII; SEQ ID NOs: 2 and 3were mixed at the same molar ratio, or siFVIII; SEQ ID NOs: 4 to 11 weremixed at the same molar ratio, or siLuc: SEQ ID NOs: 12 and 13 weremixed at the same molar ratio) 30 ug was diluted in sodium acetate (50mM) 0.75 ml to prepare an aqueous phase.

The used siRNA sequences are as follows: SEQ ID NO: 2 (FVII targetsiRNA_sense; 5′-GGAUCAUCUCAAGUCUUACdtdt-3′), SEQ ID NO: 3 (FVII targetsiRNA _antisense; 5′-GUAAGACUUGAGAUGAUCCdtdt-3′), SEQ ID NO: 4 (FVIIItarget siRNA _sense _1; 5′CUUAUAUCGUGGAGAAUUAdtdt-3′) SEQ ID NO: 5(FVIII target siRNA _antisense _1; 5′-UAAUUCUCCACGAUAUAAGdtdt-3′), SEQID NO: 6 (FVIII target siRNA sense 2; 5′-UCAAAGGAUUCGAUGGUAUdtdt-3′),SEQ ID NO: 7 (FVIII target siRNA antisense 2;5′-AUACCAUCGAAUCCUUUGAdtdt-3′), SEQ ID NO: 8 (FVIII target siRNA sense3; 5′-CAAGAGCACUAGUGAUUAUdtdt-3′), SEQ ID NO: 9 (FVIII target siRNAantisense 3; 5′-AUAAUCACUAGUGCUCUUGdtdt-3′), SEQ ID NO: 10 (FVIII targetsiRNA _sense 4; 5′-GGGCACCACUCCUGAAAUAdtdt-3′), SEQ ID NO: 11 (FVIIItarget siRNA_antisense_4; 5′-UAUUUCAGGAGUGGUGCCCdtdt-3′), SEQ ID NO: 12(siLuc_sense; 5′-AACGCUGGGCGUUAAUCAAdtdt-3′), SEQ ID NO: 13(siLuc_antisense; 5′-UUGAUUAACGCCCAGCGUUdtdt-3′).

The aqueous phase (sodium acetate or sodium citrate) in which theorganic phase (ethanol) in which the ionizable lipids, cholesterol,phospholipid and lipid-PEG conjugate (hereinafter, lipid-PEG) weredissolved and an RNA therapeutic agent (nucleic acid) were dissolvedwere mixed through a microfluid mixing device (Benchtop Nanoassemblr;PNI, Canada) at a flow rate of 12 ml/min, to prepare lipid nanoparticles(LNPs) in which the nucleic acid was encapsulated. (i) In order toprepare a lipid nanoparticle in which mRNA is encapsulated, theionizable lipid : phospholipid (DOPE) : cholesterol: lipid-PEG(C16-PEG2000 ceramide) were dissolved in ethanol at a molar ratio of26.5:20:52.5 to 51:1.0 to 2.5 (adjusting the content of cholesterol andlipid-PEG so that the total sum of the molar ratio is 100), and theorganic phase and the aqueous phase were mixed so that the mRNA(luciferase mRNA; SEQ ID NO:1):ionizable lipid was at the weight ratioof 1:10, and thereby a lipid nanoparticle was prepared. (ii) In order toprepare a lipid nanoparticle in which siRNA is encapsulated, theionizable lipid : phospholipid (DSPC) : cholesterol: lipid-PEG(C16-PEG2000 ceramide) were dissolved in ethanol at a molar ratio of42.5:13:44 to 39.5:0.5 to 5.0 (adjusting the content of cholesterol andlipid-PEG so that the total sum of the molar ratio is 100), and theorganic phase and the aqueous phase were mixed so that the siRNA(siFVII; SEQ ID NOs: 2 and 3 were mixed at the same molar ratio, orsiFVIII; SEQ ID NOs: 4 to 11 were mixed at the same molar ratio, orsiLuc: SEQ ID NOs: 12 and 13 were mixed at the same ratio): ionizablelipid was at the weight ratio of 1:7.5 and thereby a lipid nanoparticle(LNP) was prepared.

The prepared LNPs were dialyzed against PBS for 16 hours using a 3500MWCO dialysis cassette to remove ethanol and adjust the body pH and thepH of the nanoparticles.

The lipid nanoparticles comprising the ionizable lipid ‘241-C10’ werenamed ‘241-C10 LNP’, and the lipid nanoparticles prepared by using theionizable lipid comprising amine (including lipid nanoparticles in whicha nucleic acid was encapsulated) were named ‘comprised amine name (214to 246)-C10 LNP’.

Example 2-3. Observation of Nucleic Acid-Encapsulated LipidNanoparticles

The Lipid nanoparticles in which siLuc (SEQ ID NOs: 12 and 13) wereencapsulated was prepared by using a ceramide-PEG conjugate (C16-PEG2000ceramide) as Example 2-2. The prepared lipid nanoparticles (comprising1.5 mol % of ceramide-PEG conjugate) were loaded on 200 mesh carbonlacey film Cu-grid in an amount of 60 ug based on siRNA concentrationand were immersed in ethane liquefied with vitrobot (about -170 degreesor less) and were plunge frozen to be prepared, and then were observedwith Cryo-TEM (Tecnai F20, FEI), and the result was shown in FIG. 1 b .As shown in FIG. 1 b , spherical particles with a solid shape wereobserved.

Example 3. pKa of Lipid Nanoparticles

In the present example, pKa of each lipid nanoparticle (LNP) formulatedin the Example 2-1 was calculated through In vitro TNS assay. AnionicTNS becomes lipophilic by interacting with a positively chargedionizable lipid, and as the pH value becomes close to the pKa value ofeach LNP, the lipophilic property of TNS becomes lower and more watermolecules quench the TNS fluorescence, and therefore, lipidnanoparticles having a pKa of 6.0 to 7.0 have excellent in vivo drugdelivery efficiency, and lipid nanoparticles showing a “s-type curve” inthe graph representing fluorescence according to pH mean that they areeasy to interact with the endosome membrane and can easily escape theendosome during acidification.

Specifically, the pH of the solution comprising 20 mM sodium phosphate,25 mM citrate, 20 mM ammonium acetate, and 150 mM NaCl with 0.1 N NaOHand/or 0.1N HCl at an interval of 0.5 from pH 4.1 to pH 9.6 to preparesolutions of various pH units. 100 µl of each solution having each pH(pH with an interval of 0.5 from pH 4.1 to pH 9.6) was added to a black96 well plate and each was added to a solution having the pH in therange so as to be the final concentration of 6 uM using a TNS stocksolution of 300 uM. 241-C10 LNP to 246-C10 LNP were added to the mixedsolution so that the final concentration is 20 uM. The fluorescenceintensity was measured by excitation at 325 nm and emission at 435 nmthrough a Tecan equipment, and the fluorescence intensity for each lipidnanoparticle was shown in FIG. 2 and FIG. 3 , and the pKa for each lipidnanoparticle was calculated as a pH value reaching half of the maximumfluorescence and shown in Table 3 below. As shown in FIG. 3 , it couldbe seen that 244-C10 LNP to 246-C10 LNP exhibit a fluorescence titrations-shaped curve through nonlinear regression.

TABLE 3 Lipid nanoparticle pKa 241-C10 LNP 7.7 242-C10 LNP 8.7 243-C10LNP 8.2 244-C10 LNP 6.8 245-C10 LNP 6.9 246-C10 LNP 7

As confirmed in the Table 3, it was confirmed that the lipidnanoparticles according to one example showed pKa 6.0 to 7.0 range inwhich in vivo safety and drug release are excellent.

The LNPs in which a nucleic acid was encapsulated, prepared by themethod as Example 2-2, also showed the same pattern according to thetype of ionizable lipids contained (type of amine contained in theionizable lipids).

Example 4. Confirmation of Characteristics of Lipid NanoparticlesExample 4-1. Particle Size Measurement

In the present example, the size of the lipid nanoparticles (LNP;comprising 1.5 mol % of lipid-PEG) in which mRNA was encapsulatedmeasured in Example 2-2 was to be measured. It was diluted using PBS sothat the concentration of RNA (luciferase mRNA; SEQ ID NO: 1) comprisedin each lipid nanoparticle prepared in Example 2-2 was 1 ug/ml, and thediameter and polydispersity index (PDI) of the LNPs were measured usingdynamic light scattering (DLS) in Malvern Zetasizer Nano (MalvernInstruments, UK), and the result was described in Table 4 below.

TABLE 4 Lipid nanoparticle Diameter (nm) PDI 241-C10 LNP 128 0.259242-C10 LNP 77 0.210 243-C10 LNP 56 0.225 244-C10 LNP 66 0.149 245-C10LNP 70 0.210 246-C10 LNP 68 0.143

As confirmed in the Table 4, the lipid nanoparticles according to oneexample showed the particle size that is easy to be introduced intohepatocytes and has excellent drug release, and it could be found thatthe PDI values were small and the particles were uniform in order of241-C10 LNP>243-C10 LNP>242-C10 LNP=245-C10 LNP>244-C10 LNP>246-C10 LNP.

Example 4-2. Measurement of Encapsulation Efficiency

The encapsulation efficiency (drug encapsulation efficiency, %) of eachLNP (comprising 1.5 mol % of lipid-PEG) in which siRNA (siFVII siRNA)was encapsulated as a nucleic acid drug was measured through Ribogreenanalysis (Quant-iT™ RiboGreen® RNA, Invitrogen). The LNPs in which anucleic acid drug was encapsulated prepared in the Example 2-2 werediluted with lxTE buffer solution 500 in a 96 well plate so that thefinal concentration of siRNA was 4 ^(\~)7 ug/ml. To the group untreatedwith Triton-X (Triton-x LNP(-)), 1 × TE buffer 50 µl was added, and tothe group treated with Triton-X (Triton-x LNP(+)), 2% Triton-X buffer 50µl was added. By incubating at 37° C. for 10 minutes, the nucleic acidencapsulated by degrading LNPs with Triton-X was released. Then,Ribogreen reagent 100 µl was added to each well. The fluorescenceintensity (FL) of Triton LNP(-) and Triton LNP(+) was measured by thewavelength bandwidth (excitation: 485 nm, emission: 528 nm) in Infinite®200 PRO NanoQuant (Tecan), and the drug encapsulation efficiency(encapsulation efficiency, %) was calculated as the following Equation3. The drug encapsulation efficiency (%) for each LNP was shown in Table5 below as the average value of the results measured repeatedly twice.

$\begin{matrix}\begin{array}{l}{\text{Drug encapsulation efficiency (\%)=(Fluorescence intensity of Triton}} \\{\text{LNP(+)} - \text{Fluorescence intensity of Triton LNP(} - \text{))/(Fluorescence intensity of Triton}} \\{\text{LNP(+))} \times 100}\end{array} & \text{­­­(Equation 3)}\end{matrix}$

TABLE 5 Lipid nanoparticle Encapsulation efficiency (%) 241-C10 LNP 84242-C10 LNP 83 243-C10 LNP 91 244-C10 LNP 87 245-C10 LNP 91 246-C10 LNP94

As confirmed in the Table 5, it was confirmed that the lipidnanoparticles according to one example could encapsulate a drug withhigh efficiency.

Example 5. Confirmation of Intracellular Nucleic Acid Delivery UsingLipid Nanoparticles Example 5-1. Nucleic Acid Delivery Effect Accordingto Types of Ionizable Lipids Comprised in LNP

One day prior to transfection of LNP according to one example intocells, HeLa cells (Korea Cell Line Bank) were aliquoted at 0.01×10.sup.6cells/well in a white plate (96 well) and were cultured under thecondition of 37° C., 0.5^(~)3% CO.sub.2 in DMEM media (SH30022, Hyclone,USA). After stirring LNPs (241-C10 LNP to 246-C10 LNP comprising 1.5 mol% of lipid-PEG) in which mRNA (luc mRNA; SEQ ID NO: 1) encoding aluciferase gene with ApoE30.1 ug/ml by pipetting and then incubating ata room temperature for 10 minutes, they were treated (100 ng/well basedon the mRNA comprised in the lipid nanoparticles) in HeLa cells. ApoE3binds to the LNP surface and plays a role in allowing LNP to enter thecell through endocytosis through an LDL receptor expressed on the cellsurface.

In 24 hours, after treating 1000/well of Bright-Glo™ Luciferase Assaysolution (promega, USA) each and leaving them at a room temperature for10 minutes, the luminescence intensity was measured for the dissolvedcells using Infinite M200 luminescence measuring device (Tecan, USA),and the result was shown in FIG. 4 a . As shown in FIG. 4 a , 244-C10LNP, 245-C 10 LNP, and 246-C 10 LNP having a pKa range of 6.0 to 7.0showed strong luminescence intensity, and among them, 246-C10 LNP hadthe highest luminescence intensity, and therefore, it could be seen that246-C10 LNP had the highest intracellular drug delivery efficiency.

Example 5-2. Confirmation of Nucleic Acid Delivery in Hepatocytes

The luminescence intensity was measured by delivering luc mRNA intohepatocytes using 246-C10 lipid nanoparticle prepared in Example 2-2,thereby confirming expression of the gene.

Specifically, after combining 246-C10 LNP (comprising 1.5 mol % oflipid-PEG) in which luc mRNA (SEQ ID NO: 1) was encapsulated with ApoE35ug/ml, the LNP was treated into a hepatocyte cell line (Nexel, Korea)aliquoted at 1×105 cells/well at 0.2 ug/well, 0.5 ug/well, or 1 ug/wellbased on the mRNA concentration comprised in the nanoparticle. In 6hours, Bright-Glo™ Luciferase Assay solution (promega, USA) of 100µl/well was treated and left at a room temperature for 10 minutes, andthen the luminescence intensity was measured for the dissolved cellsusing Infinite M200 luminescence measuring device (Tecan, US) and theresult was shown in FIG. 4 b .

As confirmed in FIG. 4 b , it was confirmed that the lipid nanoparticleaccording to one example was easy to introduce into cells throughbinding to ApoE3, increased the amount of drug (nucleic acid) deliveryin a concentration-dependent manner, and could deliver the drug tohepatocytes with high efficiency.

Example 6. Confirmation of In Vivo Expression Using Lipid Nanoparticles

As confirmed in the Example 5-1, in vivo drug delivery efficiency andbiodistribution of 244-C10 LNP to 246-C10 LNP showing an excellent geneexpression effect (gene delivery effect) in vitro were to be confirmedin the present example.

244-C10 to 246-C10 LNP (comprising 1.5 mol % of lipid-PEG) in which lucmRNA (SEQ ID NO: 1) was encapsulated by the method of Example 2-2 wereprepared, and each nanoparticle was dialyzed in PBS for 16 hours toremove ethanol. In 3 hours after intravenously (i.v) injecting the lipidnanoparticle in which mRNA was encapsulated into C57BL/6 Female7-week-old mice (Orient Bio) in an amount of 0.1 mg/kg based on the mRNAcomprised in the lipid nanoparticle, luciferin 0.25 mg/kg wasintraperitoneally administered and the bioluminescence was confirmedthrough IVIS (PerkinElmer, USA) equipment, and the result was shown inFIG. 5 a .

Mice in which luc mRNA-encapsulated 246-C10 LNP was administered weresacrificed and organs were removed, and the biodistribution of the lipidnanoparticle was confirmed in each organ through IVIS equipment and theresult was shown in FIG. 5 b .

As shown in FIG. 5 a , mice in which luc mRNA-encapsulated 244-C10 LNPto 246-C10 LNP were administered showed high luminescence intensity, andthis corresponds to the result of the Example 5-1. In particular, asshown in FIG. 5 a and FIG. 5 b , through systemic imaging and ex vivoorgan imaging, it was confirmed that luc mRNA-encapsulated 246-C10 LNPshowed high luminescence intensity specifically to liver, and thereby itcould be confirmed that the lipid nanoparticle according to one exampleshowed high biodistribution to the liver.

Example 7. Confirmation of Composition Ratio of Lipid NanoparticlesOptimal for Nucleic Acid Delivery

In the present example, the composition ratio of the lipid nanoparticlewith the most excellent drug delivery efficiency specifically to liverin vivo was to be confirmed.

In the preparation of the lipid nanoparticle, the lipid nanoparticle(246-C10 LNP) in which luc mRNA (SEQ ID NO: 1) was encapsulated wasprepared by the method of Example 2-2 by mixing lipid-PEG (C16-PEG2000ceramide) at 1.0 to 2.5 mol %. The weight ratio of the ionizablelipid:mRNA comprised in the lipid nanoparticle was 10:1, and the molarratio of the ionizable lipid (246-C10):phospholipid(DOPE):cholesterol:lipid-PEG (C16-PEG2000 ceramide) comprised in the LNPwas 26.5:20:52.5 to 51:1.0 to 2.5 (adjusting the content of cholesteroland lipid-PEG so that the total sum of the molar ratio is 100).

For the 246-C10 LNP in which lipid-PEG was contained at 1.0 mol %, 1.5mol %, or 2.5 mol % and luc mRNA was encapsulated, similarly to themethod of Example 6, in 3 hours after mRNA-encapsulated lipidnanoparticle was intravenously (i.v) injected to C57BL/6 Female7-week-old mice (Orient Bio) at a dose of 0.1 mg/kg based on the lucmRNA contained in the lipid nanoparticle, luciferin 0.25 mg/kg wasintraperitoneally administered through IVIS (PerkinElmer, USA) equipmentto confirm bioluminescence, and the result was shown in FIG. 6 , and thesize of the lipid nanoparticles according to the lipid-PEG content wasmeasured as same as the method of Example 4-1 and was described in Table6 and FIG. 6 below.

TABLE 6 Lipid-PEG content comprised in LNP Diameter (nm) 1.0 mol% 90 1.5mol% 67 2.5 mol% 55

As shown in FIG. 6 , it could be confirmed that the group in which thelipid nanoparticle according to one example was administered hadexcellent drug delivery efficiency to the liver, and the LNP sizecomprising lipid-PEG of 1.5 mol % was about 70 nm.

Example 8. Confirmation of Hepatocyte-Specific Drug Delivery EffectExample 8-1. Confirmation of Knockout Effect of FVII Using LipidNanoparticles

FVII is expressed specifically in hepatocytes and therefore, in thepresent example, the hepatocyte targetability of the lipid nanoparticlesaccording to one example was to be confirmed through an FVII (FactorVII) knockout effect using siFVII.

So that a concentration based on the concentration of siRNA comprised inthe lipid nanoparticle was 0.03 mg/kg, 0.1 mg/kg, or 0.3 mg/kg, in 3days after the 246-C 10 lipid nanoparticle (comprising lipid-PEG of 1.5mol %) in which FVII target siRNA (SEQ ID NOs: 2 and 3) wasencapsulated, prepared in Example 2-2 was intravenously injected toC57BL/6 female 7-week-old 20 g mice, blood was collected through tailveins, and blood analysis was performed according to the protocol of thecoaset FVII assay kit, and a standard curve was drawn with blood of miceadministered with PBS and the FVII expression was measured and theresult was shown in FIG. 7 . As shown in FIG. 7 , as FVII expression wasinhibited in vivo dependently on the siRNA concentration encapsulated inthe 246-C10 lipid nanoparticle, it was confirmed that the lipidnanoparticle according to one example could deliver a nucleic acid tohepatocytes as a target.

Example 8-2. Drug Delivery Effect to Hepatocytes According to Lipid-PEGContent

The lipid nanoparticle (246-C10 LNP) in which siFVII (SEQ ID Nos: 2 and3) was encapsulated by the method of Example 2-2, by modifying thecontent of lipid-PEG comprised in the lipid nanoparticle to 0.5 to 5.0mol %, was prepared. The weight ratio of the ionizable lipid : siRNAcomprised in the lipid nanoparticle was 7.5:1, and the molar ratio ofthe ionizable lipid (246-C10) : phospholipid (DSPC) : cholesterol:lipid-PEG (C16-PEG2000 ceramide) comprised in the LNP was 42.5:13:44 to39.5:0.5 to 5.0 (adjusting the content of cholesterol and lipid-PEG sothat the total sum of the molar ratio is 100).

The diameter and polydispersity index of the lipid nanoparticlesprepared above were measured as same as the method of Example 4-1, andwere shown in Table 7 and FIG. 8 (left table) below.

TABLE 7 Lipid-PEG (%) Average diameter (nm) PDI 0.5 120 0.018 1 78 0.1061.5 52 0.159 3 42 0.152 5 37 0.226

So that a concentration based on the concentration of siRNA comprised inthe lipid nanoparticle was 0.2 mg/kg, in 3 days after the lipidnanoparticle (comprising lipid-PEG of 0.5 to 5 mol %) in which siFVIIwas encapsulated was intravenously injected to C57BL/6 female 7-week-old20 g mice, blood was collected through tail veins, and similarly to themethod of Example 8-1, using coaset FVII assay kit, the FVII expressionwas measured and the result was shown in FIG. 8 (right graph). As shownin FIG. 8 , it was confirmed that when the lipid nanoparticle accordingto one example was administered, the FVII expression in vivo wasreduced, and when the lipid nanoparticle having a lipid-PEG content of0.5 to 5.0 mol % was administered, the FVII expression was excellentlyinhibited.

Example 9. LSEC-Specific Drug Delivery Effect

As FVIII is specifically expressed in LSEC (liver sinusoidal endothelialcells), in the present example, the LSEC targetability of the lipidnanoparticle according to one example was to be confirmed through aknockout effect of FVIII (Factor VIII) using siFVIII, and the drugdelivery effect according to the lipid-PEG content was examined.

By modifying the content of lipid-PEG comprised in the lipidnanoparticle to 0.5 to 5.0 mol %, lipid nanoparticles (246-C10 LNP) inwhich siFVIII (SEQ ID Nos: 4 to 11) was encapsulated were prepared bythe method of Example 2. The weight ratio of the ionizable lipid : siRNAcomprised in the lipid nanoparticle was 7.5:1, and the ionizable lipid(246-C10) : phospholipid (DSPC) : cholesterol: lipid-PEG (C16-PEG2000ceramide) comprised in the LNP=42.5:13:44 to 39.5:0.5 to 5.0 (adjustingthe content of cholesterol and lipid-PEG so that the total sum of themolar ratio is 100).

The diameter and PDI of the lipid nanoparticles prepared above weremeasured by the same method of Example 4-1, and were shown in Table 8and FIG. 9 (left table) below.

TABLE 8 Lipid-PEG (%) Average diameter (nm) PDI 0.5 166 0.018 1 87 0.1061.5 78 0.159 3 42 0.152 5 35.6 0.226

So that a concentration based on the concentration of siRNA comprised inthe lipid nanoparticle was 0.5 mg/kg, in 2 days after the lipidnanoparticle (comprising lipid-PEG of 0.5 to 5 mol %) in which siFVIIIwas encapsulated was intravenously injected to C57BL/6 female 7-week-old20 g mice, blood was collected through tail veins, and similarly to themethod of Example 8-1, using coaset FVII assay kit, the FVIII expressionwas measured and the result was shown in FIG. 9 (right graph). As shownin FIG. 9 , it was confirmed that when the lipid nanoparticle accordingto one example was administered, the FVIII expression in vivo wasreduced, and the lipid nanoparticle according to one example couldtarget the LSEC, and when the lipid nanoparticle having a lipid-PEGcontent of 0.5 to 5.0 mol% was administered, the FVIII expression wasexcellently inhibited.

Example 10. Drug Delivery Effect According to Types of Lipid-PEGConjugates

Lipid nanoparticles (comprising a lipid-PEG conjugate of 0.25 to 10.0mol%) comprising a ceramide-PEG conjugate (C16-PEG 2000 ceramide;Avanti, US) or PEG-DSPE (Avanti, US) as a lipid-PEG conjugate wereprepared similarly to the method of Example 2-2.

The weight ratio of the ionizable lipid:siRNA (siLuc) comprised in thelipid nanoparticle was 7.5:1, and the molar ratio of the ionizable lipid(2464-C10):phospholipid (DSPC):cholesterol:lipid-PEG (ceramide-PEG orPEG-DSPE) comprised in the LNP was 42.5:13:44.25 to 34.5:0.25 to 10(adjusting the content of cholesterol and lipid-PEG so that the totalsum of the molar ratio is 100). The sequence of the used siLuc (siRNAtargeting a luciferase gene; SEQ ID Nos: 12 and 13) was described in theExample 2-2.

One day prior to transfection of LNP according to one example intocells, HeLa cells (Korea Cell Line Bank) were aliquoted at 0.01×10.sup.6cells/well in a white plate (96 well) and were cultured under thecondition of 37° C., 0.5^(~)3% CO.sub.2 in DMEM media (SH30022, Hyclone,USA). In 24 hours after treating the lipid nanoparticle in which siLucwas encapsulated to the HeLa-Luc cell line at 10 nM based on the siRNAconcentration, Bright-Glo™ Luciferase Assay solution (promega, USA) wastreated by 100 µl/well each and was left at a room temperature for 10minutes, and then for the dissolved cells, the luminescence intensitywas measured using Infinite M200 luminescence measuring device (Tecan,USA), and the result was shown in FIG. 10 . The measured result wasrepresented by mean±SD. The result value was statistically verified bythe T-test method, and a case of p<0.05 or more was defined asstatistically significant.

As shown in FIG. 10 , the lipid nanoparticle according to one examplehad an excellent nucleic acid delivery effect to cells, and inparticular, in case of comprising the ceramide-PEG conjugate as alipid-PEG conjugate, the nucleic acid delivery effect was excellent.

1. An ionizable lipid compound represented by the following formula:

.
 2. A lipid nanoparticle of claim 1 comprising the compound of claim 1.3. The lipid nanoparticle of claim 2, further comprising a phospholipid,cholesterol, and a lipid-PEG (polyethyleneglycol) conjugate.
 4. Thelipid nanoparticle according to claim 3, wherein the phospholipid is atleast one selected from the group consisting of DOPE, DSPC, POPC, EPC,DOPC, DPPC, DOPG, DPPG, DSPE, Phosphatidylethanolamine,dipalmitoylphosphatidylethanolamine,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, POPE, POPC, DOPS, and1,2-dioleoyl-sn-glycero-3-[phospho-L-serine].
 5. The lipid nanoparticleaccording to claim 3, wherein the lipid in the lipid-PEG conjugate is atleast one selected from the group consisting of ceramide,dimyristoylglycerol (DMG), succinoyl-diacylglycerol (s-DAG),distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine(DSPE), and cholesterol.
 6. The lipid nanoparticle according to claim 3,wherein the lipid-PEG conjugate is comprised in 0.25 to 10 mol%.
 7. Thelipid nanoparticle according to claim 3, which comprises the ionizablelipid, the phospholipid, the cholesterol, and the lipid-PEG conjugate ata molar ratio of 20 to 50 : 10 to 30 : 30 to 60 : 0.25 to
 10. 8. Thelipid nanoparticle according to claim 3, wherein the lipid nanoparticlehas a pKa of 6.0 to 7.0.
 9. The lipid nanoparticle according to claim 3,wherein the lipid nanoparticle specifically targets liver tissue. 10.The lipid nanoparticle according to claim 3, wherein the lipidnanoparticle targets a hepatocyte.
 11. The lipid nanoparticle accordingto claim 3, wherein the lipid nanoparticle targets an LSEC (liversinusoidal endothelial cell).
 13. A method of delivering a drug,comprising administering a composition comprising (1) the lipidnanoparticle according to of claim 3; and (2) the drug, wherein the drugis an anionic drug, a nucleic acid or a combination thereof, to asubject in need of delivering the drug.
 14. The method according toclaim 13, wherein the anionic drug, nucleic acid or combination thereofis encapsulated inside of the lipid nanoparticle.
 15. The methodaccording to claim 13, wherein the lipid nanoparticle has an averagediameter of 30 nm to 150 nm.
 16. The method according to claim 13,wherein the anionic drug is one or more kinds selected from the groupconsisting of a peptide, a drug protein, a protein-nucleic acidstructure, and an anionic biopolymer-drug conjugate.
 17. The methodaccording to claim 13, wherein the nucleic acid is one or more kindsselected from the group consisting of small interfering ribonucleic acid(siRNA), ribosome ribonucleic acid (rRNA), ribonucleic acid (RNA),deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA),aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid(tRNA), antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA andDNAzyme.
 18. A method for preventing or treating liver diseasecomprising administering a composition comprising (1) the lipidnanoparticle according to claim 1; and (2) an anionic drug, a nucleicacid or a combination thereof, to a subject in need of preventing ortreating the liver disease.
 19. The method according to claim 18,wherein the liver disease is one or more kinds selected from the groupconsisting of ATTR amyloidosis, hypercholesterolemia, hepatitis B virusinfection, acute liver failure, cirrhosis, and liver fibrosis.
 20. Themethod according to claim 18, wherein the lipid nanoparticle has anaverage diameter of 30 nm to 150 nm.